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eLife logoLink to eLife
. 2015 Jul 31;4:e07420. doi: 10.7554/eLife.07420

Registered report: Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of α-ketoglutarate-dependent dioxygenases

Brad Evans 1, Erin Griner 2; Reproducibility Project: Cancer Biology*
Editor: Irwin Davidson3
PMCID: PMC4521140  PMID: 26231040

Abstract

The Reproducibility Project: Cancer Biology seeks to address growing concerns about reproducibility in scientific research by conducting replications of selected experiments from a number of high-profile papers in the field of cancer biology. The papers, which were published between 2010 and 2012, were selected on the basis of citations and Altmetric scores (Errington et al., 2014). This Registered report describes the proposed replication plan of key experiments from ‘Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of α-ketoglutarate-dependent dioxygenases’ by Xu and colleagues, published in Cancer Cell in 2011 (Xu et al., 2011). The key experiments being replicated include Supplemental Figure 3I, which demonstrates that transfection with mutant forms of IDH1 increases levels of 2-hydroxyglutarate (2-HG), Figures 3A and 8A, which demonstrate changes in histone methylation after treatment with 2-HG, and Figures 3D and 7B, which show that mutant IDH1 can effect the same changes as treatment with excess 2-HG. The Reproducibility Project: Cancer Biology is a collaboration between the Center for Open Science and Science Exchange, and the results of the replications will be published by eLife.

DOI: http://dx.doi.org/10.7554/eLife.07420.001

Research organism: human

Introduction

Mutations in IDH1 and IDH2 are found in gliomas and in acute myeloid leukemia. All mutations are heterozygous and result in changes to one of two amino acids: arginine 132 in IDH1, or either arginine 172 or arginine 140 in IHD2. Wild-type IDH1 catalyzes the conversion of isocitrate to α-ketoglutarate (α-KG). The arginine mutations abolish its normal activity and instead mutant IDH1 and IDH2 reduce α-KG to generate the oncometabolite 2-hydroxyglutarate (2-HG) (Ward et al., 2010), which in turn affects the function of multiple α-KG dependent dioxygenases, including the TET family of 5-methylcytosine (5 mC) hydroxylases (Kinney and Pradhan, 2012; McKenney and Levine, 2013). In their Cancer Cell 2011 paper, Xu and colleagues examined the effects of excess production of 2-HG on downstream processes that could affect cancer progression. They showed that 2-HG could act as a competitive inhibitor for α-KG-dependent DNA demethylases, specifically Tet2. Ectopic expression of the mutant forms of IDH1 and IDH2 inhibited histone demethylation and 5mC hydroxylation. Examination of glioma samples from patients also showed that mutations in IDH1 were associated with increased histone methylation and decreased 5-hydroxymethylcytosine (5hmC) levels (Xu et al., 2011).

In Supplemental Figure 3I, Xu and colleagues demonstrated that transfection of U-87 MG cells with the mutant IDH1R132H increased the amount of 2-HG in the cells, as compared to transfection with wild-type IDH1 (Xu et al., 2011). This is evidence that mutant IDH1 changes the physiological levels of 2-HG, and is replicated in Protocol 1.

Xu and colleagues first showed that 2-HG can occupy the same binding pocket as α-KG in Caenorhabditis elegans KDM7A, indicating it acts as a competitive inhibitor of α-KG. Importantly, they also presented evidence that 2-HG may outcompete α-KG, since 2-HG levels affected many enzymatic functions normally dependent on α-KG. In Figure 3A, they treated U-87 MG cells with cell permeable versions of α-KG and 2-HG, and examined levels of histone methylation by Western Blot. Treatment with increasing amounts of 2-HG led to increases in H3K9me2 and H3K79me2, consistent with the idea that 2-HG inhibited histone demethylases. This effect was abolished by co-treatment with α-KG, confirming a competitive relationship between the two metabolites (Xu et al., 2011). This experiment is replicated in Protocol 2. Xu and colleagues also examined the effect of 2-HG on the TET family of 5 mC hydroxylases using an in vitro system of purified TET2 and double-stranded oligos containing a 5mC restriction digestion site in Figure 8A. Adding increasing concentrations of 2-HG abolished the ability of TET2 to convert 5 mC to 5hmC (Xu et al., 2011). This experiment will be replicated in Protocol 5.

In addition to demonstrating that the metabolite 2-HG can affect the activity of α-KG-dependent enzymes, Xu and colleagues showed that treatment with mutant forms of IDH1 and IDH2 resulted in similar outcomes. In Figure 3D, they transfected U-87 MG cells with IDH1R132H and assessed levels of histone methylation by Western blot. Transfection with IDH1R132H increased histone methylation, and treatment with α-KG abolished this increase in histone methylation, consistent with the idea that α-KG and 2-HG are competitive metabolites (Xu et al., 2011). This experiment will be replicated in Protocol 3. In Figure 7B, they also examined TET activity in the presence of mutant IDH1. While 5hmC levels are normally undetectable in HEK293 cells, transfection with TET catalytic domain (CD)-expressing plasmids increased 5hmC levels to detectable amounts. Co-transfection of TET-CD and wild-type IDH1 or IDH2 increased levels of 5hmC, as expected, while co-transfection of TET-CD with mutant forms of IDH1 and IDH2 decreased 5hmC levels (Xu et al., 2011). This experiment is replicated in Protocol 4.

The work of Xu and colleagues (Xu et al., 2011), along with work from Figueroa and colleagues (Figueroa et al., 2010) and Lu and colleagues (Lu et al., 2012), has generated much interest in the role of altered metabolites in the changing methylation patterns seen in various types of cancer. Using a different cell line than Xu and colleagues, Lu and colleagues demonstrated that mutations in IDH2, similar to mutations in IDH1, also generated abnormal levels of 2-HG which correlated with increased global methylation levels (Lu et al., 2012). Kernystsky and colleagues, Duncan and colleagues and Turcan and colleague have also shown that expression of exogenous mutated IDH genes in immortalized human cancer cell lines or in erythroid progenitor cells caused increased production of 2HG and increased levels of methylation (Duncan et al., 2012; Turcan et al., 2012; Kernytsky et al., 2015). Sasaki and colleagues extended these inquiries by generating conditional knock-in IDH1 mutant mice. These mice displayed elevated serum levels of 2HG and similar patterns of hypermethylation as observed in AML patients (Sasaki et al., 2012). Akbay and colleagues generated IDH2 mutant mice and also observed an increase in global methylation in heart tissue. They also demonstrated that mice carrying IDH mutant xenograft tumors displayed higher serum levels of 2HG (Akbay et al., 2014). Recently, 2-HG production has also been associated with MYC activation in some breast cancers, which also displayed increased levels of methylation as compared to tumors with lower levels of 2-HG (Terunuma et al., 2013).

Materials and methods

Unless otherwise noted, all protocol information was derived from the original paper, references from the original paper, or information obtained directly from the authors. An asterisk (*) indicates data or information provided by the Reproducibility Project: Cancer Biology core team. A hashtag (#) indicates information provided by the replicating lab.

Protocol 1: Gas chromatography-mass spectrometry measurement of cellular α-KG and 2-HG concentrations in U87MG cells ectopically expressing mutant IDH1

This protocol describes how to transfect cells with exogenous wild-type IDH1 or mutant IDH1R132H and assess levels of α-KH and 2-HG by gas chromatography-mass spectrometry (GC-MS), as seen in Supplemental Figure 3I.

Sampling

  • This experiment will be repeated independently 5 times for a final power of at least 92%.

    • ○ See Power calculations for details.

  • Each experiment consists of three cohorts:

    • ○ Cohort 1: U-87 MG cells transfected with vector alone.

    • ○ Cohort 1: U-87 MG cells transfected with wild-type IDH1.

    • ○ Cohort 1: U-87 MG cells transfected with mutant IDH1.

    • Each cohort will be assessed via GC–MS for:

      • ■ α-KG levels.

      • ■ 2-HG levels.

Materials and reagents

Reagent Type Manufacturer Catalog # Comments
U-87 MG cells Cells ATCC HTB-14
N-methyl-N-[tert-butyldimethylsilyl] trifluoroacetamide Chemical Sigma–Aldrich 375934-10× 1 M
Agilent 6890-5973 gas chromatograph-mass spectrometer Instrument Agilent 6890-5973
HP-5MS column Material Agilent 19091S-433I
60 mm tissue culture dishes Material Corning 430166
DMEM; high glucose Medium Sigma–Aldrich D5671 Original unspecified
Opti-MEM Reduced Serum Medium Medium Life Technologies 31985-062 Original unspecified
Vector only plasmid (GFP) Plasmid Provided by the original authors
IDH1-IRES-GFP vector Plasmid Provided by the original authors
IDH1R132H-IRES-GFP vector Plasmid Provided by the original authors
FBS Reagent Sigma–Aldrich F2442 Original unspecified
Trypsin-EDTA solution, 1× Reagent ATCC ATCC-30-2101
Penicillin-streptomycin solution Reagent ATCC ATCC-30-2300
TransIT®-LT1 transfection reagent Reagent Mirus Bio MIR 2300 Replaces SunBio-EZ (SunBio)
Methoxyamine hydrochloride Reagent Sigma–Aldrich 226904
Pyridine Reagent Sigma–Aldrich 33,553
GenElute Endotoxin-free Plasmid Maxiprep Kit Kit Sigma–Aldrich PLEX15-1KT
α-KG Chemical Sigma–Aldrich 75892
L-2-HG Chemical Sigma–Aldrich 90790
0.2 µm filter vials Material Restek 25893
Centrivap Equipment Labonco
Anti-GAPDH-HRP Antibody Abcam ab9385
Mouse monoclonal IgG1 α IDH1 Antibody Abcam ab117976 The original catalog number was not specified

Procedure

Notes
  • U-87 MG cells are maintained in DMEM supplemented with 10% FBS at 37°C/5% CO2.

  • All cells will be sent for mycoplasma testing and STR profiling.

  1. Transform, grow up and maxiprep vector only (GFP), IDH1-IRES-GFP, and IDH1R132H-IRES-GFP plasmids using a Endo-free Maxiprep kit following manufacturer's instructions.

    • a. Confirm plasmid identity by sequencing.

  2. Plate U-87 MG cells in 60 mm dishes.

    • a. First, run an optimization to determine the growth rate of the cells and optimal number of cells per plate for transfection.

  3. 24 hr after plating, transfect cells with plasmids using TransIT-LT1 transfection reagent and Opti-MEM medium according to the manufacturer's protocol.

    • a. Transfect 8 µg of DNA per construct using appropriate amount of transfection reagent.

      • i. Vector only (GFP).

      • ii. Wild-type IDH1 (IDH1-IRES-GFP).

      • iii. Mutant IDH1 (IDH1R132H-IRES-GFP).

    • b. Prepare two plates per cohort; one will be harvested for Western blot confirmation of protein expression (Step 4), the other will be used for metabolite analyses (Step 5).

  4. For Western blot: 48 hr after transfection, confirm protein expression by Western blot.

    Note: perform each time cells are transfected.

    • a. Run Western blot as outlined in Protocol 2 Steps 3 through 17 with the following modifications:

      • i. Blots do not need to be stripped and re-probed.

      • ii. Blots will be probed with:

        1. Anti-IDH1; diluted according to the manufacturer's recommendation.

        2. Anti-GAPDH-HRP; 1:5000.

          • a. Loading control.

  5. For metabolite analysis: 24 hr after transfection, remove culture medium, wash cells with cold PBS and immediately add 10 ml of pre-chilled (−80°C) 80% (vol/vol) methanol. Harvest cells by scraping and lyophilize following the manufacturer's instructions.

    • a. Samples will be lyophilized in a speedvac with no heating to keep samples frozen throughout. Immediately after drying remove samples from the speedvac for derivitization.

  6. Oximate lyophilized samples with 20 µl 20 mg/ml methoxyamine hydrochloride in pyridine at 30°C for 60 min.

  7. Derivatize samples for 30 min at 70°C in 80 μl pyridine and 20 μl N-methyl-N-[tert-butyldimethylsilyl] trifluoroacetamide.

  8. Filter samples using 0.2 µm filter vials (PTFE).

  9. Inject 3 µl of samples for gas chromatography-mass spectrometry analysis (GC–MS) into Agilent 6890-5973 GC–MS. Use a HP-5MS column (30 m 0.25 mm 0.25 μm) for analysis. Program GC oven temperature from 60°C to 180°C at 5°C/min and from 180°C to 260°C at 10°C/min. Set the flow rate of carrier gas at 1 ml/min. Operate the mass spectrometer in the electron impact (EI) mode at 70 eV.

  10. Calculate relative α-KG and 2-HG concentrations by normalizing α-KG (29.86 min) and 2-HG (30.10 min) peak areas to the average of L-threonine (29.58 min), L-serine (29.96 min) and L-phenylalanine (30.74 min) peak areas.

  11. Repeat independently four additional times.

Deliverables

  • Data to be collected:

    • ○ Chromatograms and sequence files confirming plasmid identity.

    • ○ Data generated determining growth optimization.

    • ○ Full image of Western blot showing protein expression and loading controls.

    • ○ Mass spectra readouts of all samples.

    • ○ Raw values of peak areas for α-KG (29.86 min), 2-HG (30.10 min), L-threonine (29.58 min), L-serine (29.96 min) and L-phenylalanine (30.74 min).

    • ○ Quantification of average of peak areas for L-threonine (29.58 min), L-serine (29.96 min) and L-phenylalanine (30.74 min).

    • ○ Quantification of relative α-KG and 2-HG concentrations by normalization to average peak areas for L-threonine (29.58 min), L-serine (29.96 min) and L-phenylalanine (30.74 min).

    • ○ Bar graphs of relative α-KG or 2-HG concentrations (in percent) for each cell line (as in Supplemental Figure 3I).

Confirmatory analysis plan

  • Statistical analysis of the replication data:

    • ○ Note: At the time of analysis we will perform the Shapiro–Wilk test and generate a quantile–quantile plot to assess the normality of the data. We will also perform Levene's test to assess homoscedasticity. If the data appears skewed we will perform the appropriate transformation in order to proceed with the proposed statistical analysis. If this is not possible we will perform the equivalent non-parametric test.

    • ○ One-way MANOVA of α-KG and 2-HG levels in vector-transfected, IDH1-wildtype transfected, and IDH1R132H-transfected cells with the following Bonferroni corrected comparisons:

      • ■ α-KG levels planned comparisons:

        • vector vs IDH1WT.

        • vector vs IDHR132H.

        • IDH1WT vs IDHR132H.

      • ■ 2-HG levels planned comparisons:

        • vector vs IDH1WT.

        • vector vs IDHR132H.

        • IDH1WT vs IDHR132H.

  • Meta-analysis of original and replication attempt effect sizes:

    • ○ Compute the effect sizes of each comparison, compare them against the reported effect size in the original paper and use a meta-analytic approach to combine the original and replication effects, which will be presented as a forest plot.

Known differences from the original study

  • Aspects of the Western blot protocol are provided by the replicating lab; complete details of the original protocol were unavailable.

  • Since the cell density during transfection is unknown in the original paper, the replicating lab will optimize growth conditions and cell density for transfection.

Provisions for quality control

All data obtained from the experiment—raw data, data analysis, control data and quality control data—will be made publicly available, either in the published manuscript or as an open access dataset available on the Open Science Framework (https://osf.io/kvshc/).

  • Sequence data confirming plasmid identity.

  • Western blots confirming exogenous protein expression.

  • STR profiling confirming cell line authenticity.

  • Mycoplasma testing confirming lack of contamination.

  • Growth characteristics of the cells will be optimized.

Protocol 2: Western blot to assess histone methylation in U-87 MG cells following treatment with oct-2-HG and/or oct-α-KG

This protocol describes how to treat U-87 MG cells with cell permeable versions of 2-HG and α-KG and assess histone methylation via Western blot, as seen in Figure 3A and Supplemental Figure 3F.

Sampling

  • The experiment will be repeated independently 3 times for a final power of 84%.

    • ○ See Power calculations for details.

  • Each experiment consists of four cohorts:

    • ○ Cohort 1: untreated U-87 MG cells.

    • ○ Cohort 2: U-87 MG cells treated with 10 mM racemic Oct-2-HG.

    • ○ Cohort 3: U-87 MG cells treated with 20 mM racemic Oct-2-HG.

    • ○ Cohort 4: U-87 MG cells treated with 20 mM racemic Oct-2-HG and 5 mM oct-α-KG.

    • Each sample will be blotted for:

      • ■ H3K9me2.

      • ■ H3K79me2.

      • ■ H3.

Materials and reagents

Reagent Type Manufacturer Catalog # Comments
Mouse monoclonal anti-H3K9me2 Antibody Abcam Ab1220 The original catalog number was not specified
Mouse monoclonal anti -H3K79me2 Antibody Abcam Ab3594 The original catalog number was not specified
Mouse monoclonal anti -H3 Antibody Abcam ab10799 The original catalog number was not specified
Goat Anti-Mouse IgG H&L (HRP) Antibody Abcam ab97023 We will use this for all mouse primaries
U-87 MG cells Cells ATCC HTB-14
60 mm tissue culture dishes Material Corning 430166
DMEM; high glucose Medium Sigma–Aldrich D5671 Original unspecified
FBS Reagent Sigma–Aldrich F2442 Original unspecified
Oct-α-KG Reagent Cayman Chemical 11970
2S(L)-Oct-2-HG Reagent TRC H942596 Original synthesized in house
2R(L)-Oct-2-HG Reagent TRC H942595
Protease inhibitor cocktail (mammalian) Reagent Sigma–Aldrich P8340-1ML Original not specified
TruPAGE TEA-tricine SDS running buffer (20×) Reagent Sigma–Aldrich PCG3001-500 ML Original not specified
TruPAGE LDS sample buffer (4×) Reagent Sigma–Aldrich PCG3009-10 ML Original not specified
TruPAGE DTT sample reducer (10×) Reagent Sigma–Aldrich PCG3005-1ML Original not specified
TruPAGE transfer buffer (20×) Reagent Sigma–Aldrich PCG3011-500 ML Original not specified
PBS, without MgCl2 and CaCl2 Reagent Sigma–Aldrich D8537 Original not specified
Hybond ECL nitrocellulose membranes; 20 cm × 20 cm Reagent GE Healthcare (Sigma–Aldrich) GERPN2020D Original not specified
Ponceau S solution; 0.1% (wt/vol) in 5% acetic acid Reagent Sigma–Aldrich P7170 Original not specified
Tris Buffered Saline (TBS); 10× solution Reagent Sigma–Aldrich T5912 Original not specified
Bradford reagent Reagent Sigma–Aldrich B6916 Original not specified
ECL DualVue Western Blotting Markers Reagent GE Healthcare (Sigma–Aldrich) GERPN810 Original not specified
ECL Prime Western blotting system Reagent GE Healthcare (Sigma–Aldrich) GERPN2232 Original not specified
ImageQuant Software Molecular Dynamics Version 5.2
Typhoon scanner Equipment GE Healthcare

Procedure

Notes
  • U-87 MG cells are maintained in DMEM supplemented with 10% FBS at 37°C/5% CO2.

  • All cells will be sent for mycoplasma testing and STR profiling.

  1. Plate U-87 MG cells in 60 mm dishes.

  2. 24 hr after plating, treat cells with 10 or 20 mM racemic Oct-2-HG or 5 mM Oct-α-KG or vehicle (DMSO) for 4–6 hr.

    • a. To form racemic mixtures of Oct-2-HG, mix equal amounts of the L and R enantiomers.

  3. Wash cells once with cold PBS, then lyse cells in 0.5 mL of SDS loading buffer.

    • a. 4× SDS-PAGE loading buffer: 50 mM Tris pH 6.8, 2% SDS, 10% glycerol, 1% B-ME, 12.5 mM EDTA, 0.02% bromophenol blue.

    • b. #Measure protein concentration using a CBX assay.

  4. Heat lysates at 99°C for 10 min.

  5. Run equal amounts of protein per well on a 4–20% SDS-PAGE gel at 220V until ladder marker reaches the bottom of the gel.

  6. #Equilibrate gel in transfer buffer for 15 min.

  7. #Meanwhile, cut membrane and 4 pieces 3 MM filter paper to size of gel.

    • a. Soak membrane in MeOH for a few seconds, then wash with H2O.

    • b. Soak membrane, 3 MM filter paper and pads in transfer buffer.

      • i. Transfer buffer: 38 mM glycine, 47 mM Tris, 11 mM SDS, 20% MeOH.

  8. #Assemble transfer cassette:

    • a. red pole (+) < clear plate < pad < 2 × 3 MM filter paper < membrane < gel < 2 × 3 MM filter paper < pad < black pole (−).

  9. #Add stirring bar and ice box to transfer box and fill box with transfer buffer until cassette is submerged.

    • a. Run at 100 V for 1 hr.

  10. #Wash membrane in wash buffer for 2 × 5 min.

    • a. Wash buffer: 1× PBS with 0.05% Tween-20 and 0.1% sodium azide.

  11. #Incubate membrane in blocking buffer for 30 min.

    • a. Blocking buffer: 3% non-fat milk in PBS.

  12. #Incubate membrane with one of the following primary antibody in blocking buffer for 2 hr at RT or O/N at 4°C (use manufacturer's suggested dilution in blocking buffer).

    • a. H3K9me2.

    • b. H3K79me2.

    • c. H3.

      • i. See Step 17 to strip and re-probe the blot with subsequent antibodies.

  13. #Wash 5 min 2× with wash buffer.

  14. #Incubate membrane with secondary antibody for 90 min at RT (use manufacturer's suggested dilution in blocking buffer).

    • a. HRP-conjugated Goat Anti-Mouse IgG H&L: 1: 2000.

  15. #Wash 3 × 5 min in wash buffer.

  16. #Detect HRP-conjugated secondary antibodies with chemiluminescent detection according to the manufacturer's protocol and image on the Typhoon scanner.

  17. Strip the blot in between probes:

    • a. Wash the membrane with 100 ml stripping buffer (100 mM beta-mercaptoethanol, 1% SDS 25 mM glycine pH 2.0) for 30 min with agitation.

    • b. Wash the stripped membrane twice with Western blotting wash buffer, 600 ml each wash, for 10 min with agitation.

    • c. Go to the blocking step of the western blot protocol.

    • d. Check that stripping was successful by repeating the detection step (without re-probing). Record image of the stripped gel. This will confirm the first antibody-HRP conjugate is removed and/or inactivated. If the stripping procedure is successful, wash the membrane with washing buffer and repeat the blocking-probing and detection steps for the second antibody.

      • i. Note: if stripping is unsuccessful, individual blots will be performed.

  18. Quantify intensity of bands on western blots using ImageQuant 5.2. Normalize H3k9me2 and H3K79me2 values to total H3 protein level.

  19. Repeat independently 2 additional times.

Deliverables

  • Data to be collected:

    • ○ Full scans of western blots for H3K9me2, H3K79me2 and H3 including ladder.

    • ○ Raw values of intensity of western blot bands.

    • ○ Quantification of H3K9me2 or H3K79me2 values normalized to total protein level. Levels of H3K9me2 and H3K79me2 in vehicle treated cells are set to relative intensity = 1 and all other conditions are expressed as fold change relative to the values for vehicle treated cells.

    • ○ Quantification of average values and standard deviations for each condition for triplicate experiments.

    • ○ Bar graph of average ± standard deviation of H3K9me2 and H3K79me2 levels normalized to H3 for each condition. Fold change in intensity relative to vehicle treated cells is plotted on the y axis (as seen in Supp. Figure 3F).

Confirmatory analysis plan

  • Statistical analysis of the replication data:

    • ○ Note: At the time of analysis we will perform the Shapiro–Wilk test and generate a quantile–quantile plot to assess the normality of the data. We will also perform Levene's test to assess homoscedasticity. If the data appears skewed we will perform the appropriate transformation in order to proceed with the proposed statistical analysis. If this is not possible we will perform the equivalent non-parametric test.

    • ○ One-way MANOVA of normalized H3K9me2 and H3K79me3 levels in U-87 MG cells untreated or treated with 10 mM Oct-2-HG, 20 mM Oct-2-HG, or 20 mM Oct-2-HG and 5 mM alpha-KG with the following Bonferroni corrected comparisons:

      • ■ H3K9me2 planned comparisons:

        • 0 mM 2-HG vs 10 mM 2-HG.

        • 0 mM 2-HG vs 20 mM 2-HG.

        • 20 mM 2-HG vs 5 mM α-KG + 20 mM 2-HG.

      • ■ H3K79me3 planned comparisons:

        • 0 mM 2-HG vs 10 mM 2-HG.

        • 0 mM 2-HG vs 20 mM 2-HG.

        • 20 mM 2-HG vs 5 mM α-KG + 20 mM 2-HG.

  • Additional statistical analysis for comparison to the original reported data:

    • ○ Bonferroni corrected one-sample t-tests of normalized H3K9me2 levels of the following conditions compared to 1 (0 mM 2-HG):

      • ■ 10 mM 2-HG.

      • ■ 20 mM 2-HG.

    • ○ Bonferroni corrected one-sample t-tests of normalized H3K79me3 levels of the following conditions compared to constant (0 mM 2-HG set to 1):

      • ■ 10 mM 2-HG.

      • ■ 20 mM 2-HG.

  • Meta-analysis of original and replication attempt effect sizes:

    • ○ Compute the effect sizes of each comparison, compare them against the reported effect size in the original paper and use a meta-analytic approach to combine the original and replication effects, which will be presented as a forest plot.

Known differences from the original study

  • The original racemic mixture of Oct-2-HG was synthesized in house by the original lab. The replicating lab is purchasing both L and R enantiomers and mixing them in equal amounts to form a racemic mixture.

  • Aspects of the Western blot protocol are provided by the replicating lab; complete details of the original protocol were unavailable.

Provisions for quality control

All data obtained from the experiment—raw data, data analysis, control data and quality control data—will be made publicly available, either in the published manuscript or as an open access dataset available on the Open Science Framework (https://osf.io/kvshc/).

  • STR profiling confirming cell line authenticity.

  • Mycoplasma testing confirming lack of contamination.

  • Images of stripped gel membranes confirming stripping was successful.

Protocol 3: Transfection of U-87 MG cells and determination of histone methylation by western blot

This protocol describes the transfection of U-87 MG cells with the mutant form of IDH1 and assessing methylation by Western blot, as seen in Figure 3D and Supplemental Figure 3J.

Sampling

  • This experiment will be repeated independently 6 times for a final power of 94%.

    • ○ See Power calculations for details.

  • Each experiment consists of 5 cohorts:

    • ○ Cohort 1: untransfected cells [additional control].

    • ○ Cohort 2: Vector transfected cells [additional control].

    • ○ Cohort 3: Vector transfected cells + vehicle.

    • ○ Cohort 4: IDH1R132H transfected cells + vehicle.

    • ○ Cohort 5: IDH1R132H transfected cells + 5 mM oct-α-KG.

    • ○ Each cohort is probed with antibodies against:

      • ■ H3.

      • ■ IDH1.

      • ■ H3K4me1.

      • ■ H3K4me3.

      • ■ H3K9me2.

      • ■ H3K27me2.

Materials and reagents

Reagent Type Manufacturer Catalog # Comments
Mouse monoclonal IgG3 α H3 Antibody Abcam ab10799 The original catalog number was not specified
Mouse monoclonal IgG1 α IDH1 Antibody Abcam ab117976 The original catalog number was not specified
Rabbit α H3K4me1 Antibody Abcam ab8895 The original catalog number was not specified
Mouse monoclonal IgG2b α H3K4me3 Antibody Abcam ab6000 The original catalog number was not specified
Mouse monoclonal IgG2a α H3K9me2 Antibody Abcam ab1220 The original catalog number was not specified
Rabbit α H3K27me2 Antibody Abcam ab24684 The original catalog number was not specified
Rabbit α H3K79me2 Antibody Abcam ab3594 The original catalog number was not specified
U-87 MG cells Cells ATCC HTB-14
60 mm tissue culture dishes Material Corning 430166 Or equivalent
DMEM; high glucose Medium Sigma–Aldrich D5671 Original unspecified
FBS Reagent Sigma–Aldrich F2442 Original unspecified
Empty vector plasmid Plasmid Provided by original authors
IDH1R132H expression vector Plasmid Provided by original authors
TransIT®-LT1 transfection reagent Reagent Mirus Bio MIR 2300 Replaces SunBio-EZ (SunBio)
Oct-α-KG Reagent Cayman Chemical 11970
Typhoon scanner Equipment GE Healthcare
ImageQuant Software Molecular Dynamics Version 5.2
Protease inhibitor cocktail (mammalian) Reagent Sigma–Aldrich P8340-1ML Original not specified
TruPAGE TEA-tricine SDS running buffer (20×) Reagent Sigma–Aldrich PCG3001-500 ML Original not specified
TruPAGE LDS sample buffer (4×) Reagent Sigma–Aldrich PCG3009-10 ML Original not specified
TruPAGE DTT sample reducer (10×) Reagent Sigma–Aldrich PCG3005-1ML Original not specified
TruPAGE transfer buffer (20×) Reagent Sigma–Aldrich PCG3011-500 ML Original not specified
PBS, without MgCl2 and CaCl2 Reagent Sigma–Aldrich D8537 Original not specified
Hybond ECL nitrocellulose membranes; 20 cm × 20 cm Reagent GE Healthcare (Sigma–Aldrich) GERPN2020D Original not specified
Ponceau S solution; 0.1% (wt/vol) in 5% acetic acid Reagent Sigma–Aldrich P7170 Original not specified
Tris buffered saline (TBS); 10× solution Reagent Sigma–Aldrich T5912 Original not specified
Bradford reagent Reagent Sigma–Aldrich B6916 Original not specified
ECL DualVue Western blotting markers Reagent GE Healthcare (Sigma–Aldrich) GERPN810 Original not specified
ECL prime Western blotting system Reagent GE Healthcare (Sigma–Aldrich) GERPN2232 Original not specified
Goat Anti-Rabbit IgG H&L (HRP) Antibody Abcam ab97051
Goat Anti-Mouse IgG H&L (HRP) Antibody Abcam ab97023

Procedure

Notes
  • U-87 MG cells are maintained in DMEM supplemented with 10% FBS at 37°C/5% CO2.

  • All cells will be sent for mycoplasma testing and STR profiling.

  1. Plate U-87 MG cells in 60 mm dishes.

  2. 24 hr after plating, transfect cells with plasmids (maxiprepped in Protocol 1) using TransIT-LT1 Transfection Reagent according to manufacturer's protocol.

    • a. #Transfect 8 µg of DNA per construct using appropriate volume of transfection reagent.

      • i. Empty vector.

      • ii. IDH1R132H vector.

  3. 48 hr after transfection, treat cells with vehicle or 5 mM Oct-α-KG for 6 hr.

    • a. Vehicle is DMSO.

  4. Wash cells once with cold PBS, then lyse cells in 0.5 ml of SDS loading buffer.

    • a. #4 SDS-PAGE loading buffer: 50 mM Tris pH 6.8, 2% SDS, 10% glycerol, 1% B-ME, 12.5 mM EDTA, 0.02% bromophenol blue.

  5. Heat lysates at 99°C for 10 min.

  6. Run SDS-PAGE gel until ladder marker reaches the bottom of the gel.

  7. #Equilibrate gel in transfer buffer for 15 min.

  8. #Meanwhile, cut membrane and 4 pieces 3 MM filter paper to size of gel.

    • a. Soak membrane in MeOH for a few seconds, then wash with H2O.

    • b. Soak membrane, 3 MM filter paper and pads in transfer buffer.

      • i. Transfer buffer: 38 mM glycine, 47 mM Tris, 11 mM SDS, 20% MeOH.

  9. #Assemble transfer cassette:

    • a. red pole (+) < clear plate < pad < 2 × 3 MM filter paper < membrane < gel < 2 × 3 MM filter paper < pad < black pole (−).

  10. #Add stirring bar and ice box to transfer box and fill box with transfer buffer until cassette is submerged.

    • a. Run at 100 V for 1 hr.

  11. #Wash membrane in wash buffer for 2 × 5 min.

    • a. Wash buffer: 1× PBS with 0.05% Tween-20 and 0.1% sodium azide.

  12. #Incubate membrane in blocking buffer for 30 min.

    • a. Blocking buffer: 3% non-fat milk in PBS.

  13. #Incubate membrane with primary antibody in blocking buffer for 2 hr at room temperature (RT) or overnight at 4°C (use manufacturer's suggested dilution in blocking buffer).

    • a. H3.

    • b. IDH1.

    • c. H3K4me1.

    • d. H3K4me3.

    • e. H3K9me2.

    • f. H3K27me2.

    • g. H3K79me2.

  14. #Wash 5 min 2× with wash buffer.

  15. #Incubate membrane with secondary antibody for 90 min at RT (use manufacturer's suggested dilution in blocking buffer).

    • a. HRP-conjugated Goat Anti-Mouse IgG H&L: 1:2000.

    • b. HRP-conjugated Goat Anti-Rabbit IgG H&L: 1:2000.

  16. #Wash 3 × 5 min in wash buffer.

  17. # Detect HRP-conjugated secondary antibodies with chemiluminescent detection according to the manufacturer's protocol and image on the Typhoon scanner.

  18. Strip the blot in between probes:

    • a. Wash the membrane with 100 ml stripping buffer (100 mM betamercaptoethanol, 1% SDS 25 mM glycine pH 2.0) for 30 min with agitation.

    • b. Wash the stripped membrane twice with Western blotting wash buffer, 600 ml each wash, for 10 min with agitation.

    • c. Go to the blocking step of the western blot protocol.

    • d. Check that stripping was successful by repeating the detection step (without re-probing). Record image of the stripped gel. This will confirm the first antibody-HRP conjugate is removed and/or inactivated. If the stripping procedure is successful, wash the membrane with washing buffer and repeat the blocking-probing and detection steps for the second antibody.

      • i. Note: if stripping is unsuccessful, individual blots will be performed.

  19. Quantify intensity of bands on western blots using ImageQuant 5.2. Normalize levels of methylated histones to total H3 protein level. Normalize IDH1R132H + vehicle and IDH1R132H + oct-α-KG treated samples to vector + vehicle samples for each normalized methylated histone.

  20. Repeat independently 5 additional times.

Deliverables

  • Data to be collected:

    • ○ Full scans of western blots for H3, IDH1, H3K4me1, H3K4me3, H3K9me2, H3K27me2, and H3K79me2 (as seen in Figure 3D) including ladder.

    • ○ Raw values of intensity of western blot bands as measured by ImageQuant 5.2 software.

    • ○ Quantification of methylated histone values normalized to total protein level.

    • ○ Quantification of average values and standard deviations for each condition. Levels of methylated histone in vector control cells are set to 100% and levels of methylated histone for other conditions are relative to vector control.

    • ○ Table of average ± standard deviation of methylated histone levels normalized to H3 for each condition and relative to vector control cells (as seen in Supplemental Figure 3J).

Confirmatory analysis plan

  • Statistical analysis of the replication data:

    • ○ Note: At the time of analysis we will perform the Shapiro–Wilk test and generate a quantile–quantile plot to assess the normality of the data. We will also perform Levene's test to assess homoscedasticity. If the data appears skewed we will perform the appropriate transformation in order to proceed with the proposed statistical analysis. If this is not possible we will perform the equivalent non-parametric test.

    • ○ One-way MANOVA of normalized H3K4me1, H3K4me3, H3K9me2, H3K27me2, and H3K79me2 levels from IDH1R132H + vehicle and IDH1R132H + oct-α-KG cells with the following Bonferroni corrected comparisons:

      • ■ H3K4me1 levels of IDH1R132H vs IDH1R132H + oct-α-KG.

      • ■ H3K4me3 levels of IDH1R132H vs IDH1R132H + oct-α-KG.

      • ■ H3K9me2 levels of IDH1R132H vs IDH1R132H + oct-α-KG.

      • ■ H3K27me2 levels of IDH1R132H vs IDH1R132H + oct-α-KG.

      • ■ H3K79me2 levels of IDH1R132H vs IDH1R132H + oct-α-KG.

    • ○ Bonferroni corrected one-sample t-tests (outside the MANOVA framework) of normalized levels from IDH1R132H + vehicle of the following conditions compared to constant (vector + vehicle set to 100):

      • ○ H3K4me1.

      • ○ H3K4me3.

      • ○ H3K9me2.

      • ○ H3K27me2.

      • ○ H3K79me2.

  • Meta-analysis of original and replication attempt effect sizes:

    • ○ Compute the effect sizes of each comparison, compare them against the reported effect size in the original paper and use a meta-analytic approach to combine the original and replication effects, which will be presented as a forest plot.

Known differences from the original study

  • While the manufacturer was specified for antibodies used, the exact catalog number was not. The RP:CB core team chose the most appropriate antibody from the manufacturer based on manufacturer's recommended applications and user reviews of the antibody.

  • Aspects of the Western blot protocol are provided by the replicating lab; complete details of the original protocol were unavailable.

Provisions for quality control

All data obtained from the experiment—raw data, data analysis, control data and quality control data—will be made publicly available, either in the published manuscript or as an open access dataset available on the Open Science Framework (https://osf.io/kvshc/).

  • STR profiling confirming cell line authenticity.

  • Mycoplasma testing confirming lack of contamination.

  • Images of stripped gel membranes confirming stripping was successful.

Protocol 4: Dot blot to measure of levels of 5hmC in genomic DNA

This protocol describes how to transfect HEK293 cells with vectors expressing the catalytic domain of TET2 (TET2-CD) and wild-type or mutant forms of IHD1 and IDH2 and then assess genomic DNA hydroxymethylation by dot blot, as seen in Figure 7B and Supplemental Figure 7C.

Sampling

  • This experiment will be conducted independently 4 times for a final power of 96%.

    • ○ See Power calculations for details.

  • Each experiment consists of 9 cohorts:

    • ○ Cohort 1: Untransfected cells [additional control].

    • ○ Cohort 2: Vector transfected cells.

    • ○ Cohort 3: FLAG-TET2-CD transfected cells.

      • ■ The catalytic domain of TET2.

    • ○ Cohort 4: FLAG-TET2-CM transfected cells.

      • ■ CM: mutant version of the TET2 catalytic domain.

    • ○ Cohort 5: FLAG-TET2-CD + FLAG-IDH1 transfected cells.

    • ○ Cohort 6: FLAG-TET2-CD + FLAG-IDH1R132H transfected cells.

    • ○ Cohort 7: FLAG-TET2-CD + FLAG-IDH2 transfected cells.

    • ○ Cohort 8: FLAG-TET2-CD + FLAG-IDH2R140Q transfected cells.

    • ○ Cohort 9: FLAG-TET2-CD + FLAG-IDH2R172K transfected cells.

    • ○ Each cohort will have gDNA spotted out at 5, 10, 25, 50, 100 and 250 ng and probed with anti-5hmC antibody.

Materials and reagents

Reagent Type Manufacturer Catalog # Comments
Mouse monoclonal IgG2a α Anti-5hmC Antibody Active Motif 40000 Original catalog number unspecified
Mouse monoclonal IgG1 α FLAG Antibody Sigma–Aldrich F3165 Original catalog number unspecified
Goat Anti-Mouse IgG H&L (HRP) Antibody Abcam ab97023
HEK293 cells Cells ATCC CRL-1573 Original unspecified
Typhoon scanner Equipment Amersham/ GE Health Sciences 9410
Hybond ECL nitrocellulose membranes; 20 cm × 20 cm Reagent GE Healthcare (Sigma–Aldrich) GERPN2020D Original not specified
DMEM; high glucose Medium Sigma–Aldrich D5671 Original unspecified
FBS Reagent Sigma–Aldrich F2442 Original unspecified
Vector alone Plasmid Provided by original authors
FLAG-TET2-CD Plasmid Provided by original authors
FLAG-TET2-CM Plasmid Provided by original authors
FLAG-IDH1 Plasmid Provided by original authors
FLAG-IDH1R132H Plasmid Provided by original authors
Flag-IDH2 Plasmid Provided by original authors
FLAG-IDH2R140Q Plasmid Provided by original authors
FLAG-IDH2R172K Plasmid Provided by original authors
TransIT-LT1 transfection reagent Reagent Mirus Bio MIR 2300 Replaces SunBio-EZ (SunBio)
Nonfat-dried milk bovine Reagent Sigma–Aldrich M7409
ECL prime Western blotting system Reagent GE Healthcare (Sigma–Aldrich) GERPN2232 Original not specified
Image Quant 5.2 Software GE Version 5.2
Protease inhibitor cocktail (mammalian) Reagent Sigma–Aldrich P8340-1ML Original not specified
TruPAGE TEA-Tricine SDS running buffer (20×) Reagent Sigma–Aldrich PCG3001-500 ML Original not specified
TruPAGE LDS sample buffer (4×) Reagent Sigma–Aldrich PCG3009-10 ML Original not specified
TruPAGE DTT sample reducer (10×) Reagent Sigma–Aldrich PCG3005-1ML Original not specified
TruPAGE transfer buffer (20×) Reagent Sigma–Aldrich PCG3011-500 ML Original not specified
PBS, without MgCl2 and CaCl2 Reagent Sigma–Aldrich D8537 Original not specified
Ponceau S solution; 0.1% (wt/vol) in 5% acetic acid Reagent Sigma–Aldrich P7170 Original not specified
Tris buffered saline (TBS); 10× solution Reagent Sigma–Aldrich T5912 Original not specified
Bradford reagent Reagent Sigma–Aldrich B6916 Original not specified
QIAamp DNA mini kit Kit Qiagen 51304

Procedure

Notes
  • This protocol contains information from Ito and colleagues (Ito et al., 2010).

  • HEK293 cells are maintained in DMEM supplemented with 10% FBS at 37°C/5% CO2.

  • All cells will be sent for mycoplasma testing and STR profiling.

  1. Transform, grow up and maxiprep plasmids using an Endo-free Maxiprep kit following the manufacturer's instructions.

    • a. Confirm plasmid identity by sequencing.

  2. Plate 6 × 105 − 1.2 × 106 HEK293 cells per 60 mm dish.

  3. 24 hr after plating, transfect cells with indicated plasmids.

    • a. #Transfect cells with 8 µg of DNA per construct using TransIT-LT1 Transfection Reagent according to manufacturer's protocol#.

      • i. Cohort 1: Untransfected cells.

      • ii. Cohort 2: Vector only.

      • iii. Cohort 3: FLAG-TET2-CD.

      • iv. Cohort 4: FLAG-TET2-CM.

      • v. Cohort 5: FLAG-TET2-CD + FLAG-IDH1.

      • vi. Cohort 6: FLAG-TET2-CD + FLAG-IDH1R132H.

      • vii. Cohort 7: FLAG-TET2-CD + FLAG-IDH2.

      • viii. Cohort 8: FLAG-TET2-CD + FLAG-IDH2R140Q.

      • ix. Cohort 9: FLAG-TET2-CD + FLAG-IDH2R172K.

  4. *For each cohort, transfect two parallel plates; harvest genomic DNA from one plate (proceed to Step 5) and protein from the second plate (proceed to Step 7).

  5. 36–40 hr after transfection, isolate genomic DNA from cells on the first plate using the QIAamp kit according to the manufacturer's instructions.

    • a. Determine DNA concentration and purity.

  6. Dot blot to assess levels of 5hmC:

    • a. Quantify gDNA concentration using a NanoDrop. #Spot genomic DNA onto nitrocellulose membrane using a pipet, then crosslink the DNA to the membrane by UV irradiation for 2 min.

      • i. The following amounts of genomic DNA should be spotted: 250 ng, 100 ng, 50 ng, 25 ng, 10 ng, and 5 ng.

    • b. Bake nitrocellulose membrane at 80°C for #1 hr.

    • c. Block membrane with 5% skim milk in TBS with 0.1% Tween 20 (TBST) for 1 hr.

    • d. Perform western blot on spotted nitrocellulose with the following antibody: anti-5hmC. Incubate membrane with primary antibody diluted 1:10,000 overnight at 4°C.

    • e. Wash membrane three times with TBST.

    • f. Incubate membrane with secondary antibody (HRP-conjugated anti-rabbit IgG) diluted 1:2000 for 1 hr at room temperature.

    • g. Wash membrane three times with TBST, then treat with ECL and scan with a Typhoon scanner.

    • h. Quantify dot-blot using Image-Quanta software.

  7. Check expression of exogenous proteins by Western blot using the second plate.

    • a. Wash cells once with cold PBS, then lyse cells in 0.5 ml of SDS loading buffer.

      • i. #4× SDS-PAGE loading buffer: 50 mM Tris pH 6.8, 2% SDS, 10% glycerol, 1% B-ME, 12.5 mM EDTA, 0.02% bromophenol blue.

    • b. Heat lysates at 99°C for 10 min.

    • c. Run SDS-PAGE gel until ladder marker reaches the bottom of the gel.

    • d. #Equilibrate gel in transfer buffer for 15 min.

    • e. #Meanwhile, cut membrane and 4 pieces 3 MM filter paper to size of gel.

      • i. Soak membrane in MeOH for a few seconds, then wash with H2O.

      • ii. Soak membrane, 3 mM filter paper and pads in transfer buffer.

      • iii. Transfer buffer: 38 mM glycine, 47 mM Tris, 11 mM SDS, 20% MeOH

    • f. #Assemble transfer cassette:

      • i. red pole (+) < clear plate < pad < 2 × 3 MM filter paper < membrane < gel < 2 × 3 MM filter paper < pad < black pole (−).

    • g. #Add stirring bar and ice box to transfer box and fill box with transfer buffer until cassette is submerged.

      • i. Run at 100 V for 1 hr.

    • h. #Wash membrane in wash buffer for 2 × 5 min.

      • i. Wash buffer: 1× PBS with 0.05% Tween-20 and 0.1% sodium azide.

    • i. #Incubate membrane in blocking buffer for 30 min.

      • i. Blocking buffer: 3% non-fat milk in PBS.

    • j. #Incubate membrane with primary antibody in blocking buffer for 2 hr at RT or O/N at 4°C (use manufacturer's suggested dilution in blocking buffer).

      • i. α FLAG.

    • k. #Wash 5 min 2× with wash buffer.

    • l. #Incubate membrane with secondary antibody for 90 min at RT (use manufacturer's suggested dilution in blocking buffer).

      • i. HRP-conjugated Goat Anti-Mouse IgG H&L: 1:2000.

    • m. #Wash 3 × 5 min in wash buffer.

    • n. # Detect HRP-conjugated secondary antibodies with chemiluminescent detection according to the manufacturer's protocol and image on the Typhoon scanner.

    • o. Quantify intensity of dots on western blots using ImageQuant 5.2.

      • i. Normalize values to FLAG-TET2-CD transfected cells.

  8. Repeat independently three additional times.

Deliverables

  • Data to be collected:

    • ○ Chromatograms and sequence files confirming plasmid identity.

    • ○ DNA concentration and purity data.

    • ○ Full scans of dot blots for anti-5hmC and western blots for anti-FLAG (as seen in Figure 7B).

    • ○ Raw values of intensity of dot blot as measured by Image-Quanta software.

    • ○ Quantification of 5hmc values relative to TET2-CD.

    • ○ Quantification of average values and standard deviations for each condition for all experiments.

    • ○ Bar graph and table of average values and standard deviations relative to TET2-CD samples (as seen in Figure 7B and Supplemental Figure 7C).

Confirmatory analysis plan

  • Statistical analysis of the replication data:

    • ○ Note: At the time of analysis we will perform the Shapiro–Wilk test and generate a quantile–quantile plot to assess the normality of the data. We will also perform Levene's test to assess homoscedasticity. If the data appears skewed we will perform the appropriate transformation in order to proceed with the proposed statistical analysis. If this is not possible we will perform the equivalent non-parametric test.

    • ○ Comparison of the various genotypes for each of the DNA concentrations.

      • ■ Bonferonni corrected one-sample t-test of normalized 5hmC levels of the following cohorts compared to constant (TET2-CD set to 1):

        • TET-2CD + IDH1.

        • TET-2CD + IDH1R132H.

        • TET-2CD + IDH2.

        • TET-2CD + IDH2R140Q.

        • TET-2CD + IDH2R172K.

  • Meta-analysis of original and replication attempt effect sizes:

    • ○ Compute the effect sizes of each comparison, compare them against the reported effect size in the original paper and use a meta-analytic approach to combine the original and replication effects, which will be presented as a forest plot.

Known differences from the original study

  • Aspects of the Western blot protocol are provided by the replicating lab; complete details of the original protocol were unavailable.

Provisions for quality control

All data obtained from the experiment—raw data, data analysis, control data and quality control data—will be made publicly available, either in the published manuscript or as an open access dataset available on the Open Science Framework (https://osf.io/kvshc/).

  • Sequence data confirming plasmid identity.

  • Western blots confirming exogenous protein expression.

  • STR profiling confirming cell line authenticity.

  • Mycoplasma testing confirming lack of contamination.

Protocol 5: Radiolabeled 5mC-5hmC conversion assay

This protocol describes how to run the in vitro assay to examine the effect of 2-HG on the TET family of methyl hydroxylases, as seen in Figure 8A.

Sampling

  • This experiment will be performed independently a total of 6 times for a final power of ≥80%.

    • ○ The original data is qualitative, thus to determine an appropriate number of replicates to initially perform, sample sizes based on a range of potential variance was determined.

    • ○ See Power calculations for details.

  • Each experiment consists of 8 cohorts:

    • ○ No recombinant protein.

    • ○ FLAG-TET2-CD + vehicle.

    • ○ FLAG-TET2-CD + 10 mM D-2-HG.

    • ○ FLAG-TET2-CD + 25 mM D-2-HG.

    • ○ FLAG-TET2-CD + 50 mM D-2-HG.

    • ○ FLAG-TET2-CD + 10 mM L-2-HG.

    • ○ FLAG-TET2-CD + 25 mM L-2-HG.

    • ○ FLAG-TET2-CD + 50 mM L-2-HG.

    • ○ Each cohort will detect:

      • ■ 5m-dCMP.

      • ■ 5hm-dCMP.

Materials and reagents

Reagent Type Manufacturer Catalog # Comments
D-2-HG Reagent Sigma–Aldrich H8378
L-2-HG Reagent Sigma–Aldrich 90790
Sf9 cells Cells ATCC CRL-1711 Original unspecified
Shrimp alkaline phosphatase Reagent New England Biolabs MO371S
T4 polynucleotide kinase Reagent Sigma–Aldrich KEM0006
DNase I Reagent Sigma–Aldrich AMPD1
Phosphodiesterase I Reagent Sigma–Aldrich P3243
PEI-cellulose TLC plate Material Sigma–Aldrich Z122882
FLAG-TET2-CD viral particles Virus Provided by the original authors
Anti-Flag M2 antibody agarose affinity gel Reagent Sigma–Aldrich A2220
Flag peptide Reagent Sigma–Aldrich F4799
α-KG Reagent Sigma–Aldrich 75,892
GenElute PCR Clean-Up Kit Kit Sigma–Aldrich NA1020-1KT Replaces Qiagen cat no. 28304
[γ-32]ATP Reagent Perkin Elmer BLU502H/NEG502H
MspI methyltransferase Reagent NEB M0215L
MspI restriction endonuclease Reagent NEB R0106T
DNA duplex oligonucleotide substrate oligo Integrated DNA Technologies custom 5′-GTGTTCTTTCAGCTCCGGTCACGCTGACCAGC-3′ as a duplex oligo, HPLC purified at 1 umole scale maybe higher depending on recovery
M13-F primer oligo Integrated DNA Technologies CCAGTCACGACGTTGTAAAACG
M13-R primer oligo Integrated DNA Technologies CCAGTCACGACGTTGTAAAACG
JumpStart REDTaq DNA Polymerase Reagent Sigma D8189-50UN
dNTP mix 10 mM Reagent Sigma D7295-.2 ML
BlueView TAE buffer Buffer Sigma T8935-1L
Molecular biology grade water Reagent Sigma W4502-1L

Procedure

Note: This protocol contains information from Ito and colleagues (2010).

  1. Generate recombinant FLAG-TET2-CD virus from supplied virus stock.

    • a. #Infect a 5 ml culture with 0.1 ml of virus stock supplied.

      • i. Grow in a stationary tissue culture flask at 27°C.

    • b. #After 5 days, collect the virus. Simultaneously, start a 50 ml suspension culture at 27°C with 140 rpm shaking.

      • i. Confirm viral insert identity by sequencing using M13F and R primers and REDTaq polymerase, followed by gel purification and sequencing of PCR product.

    • c. #After culturing for 3 days, infected the suspension culture with 2.5 ml of virus stock.

    • d. #4 days after infection collect virus. Simultaneously, start new 50 ml suspension cultures for protein expression.

      • e. #After 3 days of culture, the suspension cultures are infected with 2.5 ml virus.

    • e. #After 3 days of infection the cells expressing recombinant protein are collected by centrifugation and stored at −80°C until the protein is to be purified.

      • i. #More round of expression may be required depending on expression level.

    • f. Purify baculovirus expressed recombinant FLAG-TET2-CD from insect Sf9 cells with anti-Flag M2 antibody agarose affinity gel and elute with buffer containing 10 mM Tris–HCl pH 8.0, 150 mM NaCl, 1 mM DTT, 15% glycerol and 0.2 μg/µl Flag peptide.

    • g. Note; generate sufficient recombinant protein to use in a total of 6 replicates of this protocol.

  2. #Prepare methylated oligonucleotide substrate.

    • a. Treat unmethylated DNA duplex oligo with MspI methyltransferase for 2 hr at 37°C following manufacturer's instructions.

    • b. Purify with a QiaQuick Nucleotide Removal kit following manufacturer's instructions.

  3. Incubate 5 µg of the purified recombinant TET2-CD protein and various concentrations of vehicle only, D-2-HG, or L-2-HG with 0.5 μg methylated oligonucleotide substrate in vehicle (50 mM HEPES (pH 8), 75 μM Fe(NH4)2(SO4)2, 2 mM ascorbate) and 0.1 mM α-KG for 3 hr at 37°C.

    • a. See cohorts for detailed concentrations to use.

    • b. #If necessary, concentrate protein to ensure the final reaction volume is between 100–1000 µl.

    • c. Purify oligonucleotide substrates using a GenElute PCR Clean-Up Kit following manufacture's instructions.

  4. Digest oligonucleotides with 1 U/μg MspI restriction endonuclease at 37°C for #2 hr following manufacturer's instructions.

  5. Treat digested DNA with 1U/μmol shrimp alkaline phosphatase at 37°C for #2 hr.

    • a. #Heat inactivate at 65°C for 10 min.

  6. Label DNA with [γ-32]ATP and polynucleotide kinase.

    • a. #Add 1 µl of [γ-32]ATP at 3000 Ci/mmol, 5 mCi/ml and 1 µl polynucleotide kinase to the previous reaction.

    • b. #Incubate for 1 hr at 37°C.

  7. Ethanol precipitate labeled fragments.

    • i. #Add 3M NaOAc to a final concentration of 0.3M.

    • ii. #Add 2 vol 100% EtOH.

    • iii. #Incubate mixture at on dry ice for 20 min.

    • iv. #Centrifuge in a microfuge at 4°C at maximum speed for 10 min.

    • v. #Remove supernatant and air dry pellet.

    • vi.#Resuspend.

  8. Digest labeled fragments with 10 µg DNAse I and 10 μg phosphodiesterase I in the presence of 15 mM MgCl2 and 2 mM CaCl2 at 37 °C for #2 hr.

  9. Spot 1 µl of digestion product from step 8 onto a PEI-cellulose TLC plate and separate in an isobutyric acid/water/ammonium hydroxide (66:20:2) buffer.

  10. Dry the TLC plate and then expose to film.

  11. Quantify intensity of 5hmC bands.

    • a. Normalize values to FLAG-TET2-CD + vehicle.

  12. Repeat independently five additional times starting at Step 2.

Deliverables

  • Data to be collected:

    • ○ Sequencing data confirming viral insert identity.

    • ○ Data about viral titer and amount of and quality of protein generated.

    • ○ Scans of films exposed to TLC plate (as in Figure 8A, left).

    • ○ Raw values of intensity of 5hm-dCMP (5hmC) spots.

    • ○ Quantification of 5hmC intensity relative to FLAG-TET2-CD (recombinant protein) + vehicle sample.

    • ○ Quantification of average values and standard deviations for each condition for triplicate experiments.

    • ○ Bar graph of relative 5hmC intensity for each sample with standard deviations (as in Figure 8A, right).

Confirmatory analysis plan

  • Statistical Analysis of the Replication Data:

    • ○ Note: At the time of analysis we will perform the Shapiro–Wilk test and generate a quantile–quantile plot to assess the normality of the data. We will also perform Levene's test to assess homoscedasticity. If the data appears skewed we will perform the appropriate transformation in order to proceed with the proposed statistical analysis. If this is not possible we will perform the equivalent non-parametric test.

    • ○ Two-way ANOVA of normalized 5hmC levels of TET2-CD protein treated with D-2-HG or L-2-HG with the following Bonferroni corrected comparisons:

      • 10 mM D-2-HG vs 10 mM L-2-HG.

      • 50 mM D-2-HG vs 50 mM L-2-HG.

      • 10 mM D-2-HG vs 50 mM D-2-HG.

    • ○ Bonferroni corrected one-sample t-tests (outside the ANOVA framework) of normalized 5hmC levels of TET2-CD protein treated with the following concentrations of D-2-HG compared to constant (TET2-CD + vehicle set to 1):

      • 10 mM D-2-HG.

      • 50 mM D-2-HG.

  • Meta-analysis of original and replication attempt effect sizes:

    • ○ The replication data (mean and 95% confidence interval) will be plotted with the original reported data value plotted as a single point on the same plot for comparison.

Known differences from the original study

  • The lab provided the protocol for expansion of the viral aliquot shared by the original authors for generation of the recombinant FLAG-TET2 protein.

Provisions for quality control

All data obtained from the experiment—raw data, data analysis, control data and quality control data—will be made publicly available, either in the published manuscript or as an open access dataset available on the Open Science Framework (https://osf.io/kvshc/).

  • Sequence data confirming viral insert identity.

  • Data about viral titer and amount of and quality of protein generated.

Power calculations

Power calculations are performed to calculate the number of samples required to achieve at least 80% power and the indicated alpha error. For a detailed breakdown of all power calculations, please see spreadsheet at https://osf.io/gnsti/wiki/home/.

Protocol 1

Summary of original data
  • Note: Data estimated from published figures.

Supp. Figure 3I: Levels of α-KG with WT or mutant IDH1 Mean SD N
Vector-transfected U-87 MG cells 84 0* 2
WT IDH-transfected U-87 MG cells 120 7.8 2
IDHR132H-transfected U-87 MG cells 41 14 2
Supp. Figure 3I: Levels of 2-HG with WT or mutant IDH1. Mean SD N
Vector-transfected U-87 MG cells 90 0* 2
WT IDH-transfected U-87 MG cells 140 0* 2
Mutant IDH-transfected U-87 MG cells 1730 14 2
*

Because the original data reported null variances, the calculations below used the average of the non-null variances, 11.9, in place of a SD of 0.

Test family
  • Due to a lack of raw original data, we are unable to perform power calculations using a MANOVA. We are determining sample size calculations using a two-way ANOVA.

  • Two-way ANOVA followed by Bonferroni corrected comparisons.

Power calculations
ANOVA calculations; α = 0.05
F(1,6) metabolite Partial η2 Effect size f Power Total sample size
6702.3 0.999106 33.43005 99.9% 7*
*

With 5 samples per group (30 samples total), power achieved is 99.9%.

Corrected t-test sample size calculations; α = 0.0083333
Group 1 Group 2 Effect size d Power Sample size per group
α-KG Vector IDH1-WT 3.57815 80.1%* 4*
Vector IDH1-R132H 3.30960 92.0% 5
IDH1-WT IDH1-R132H 6.97125 97.4% 3
2HG Vector IDH1-WT 4.20168 93.1% 4
Vector IDH1-R132H 126.22669 99.9%§ 2§
IDH1-WT IDH1-R132H 122.37832 99.9%# 2#
*

With a sample size of 5 per group, the achieved power is 95.7%.

With a sample size of 5 per group, the achieved power is 99.9%.

With a sample size of 5 per group, the achieved power is 99.2%.

§

With a sample size of 5 per group, the achieved power is 99.9%.

#

With a sample size of 5 per group, the achieved power is 99.9%.

Sensitivity calculations
  • Comparing 2-HG levels from Vector to IDH1 WT:

    • ○ Based on a sample size of 4 per group, we will be able to see an effect size of 3.3710662 with α = 0.01 and a power of 80%.

Protocol 2

Summary of original data
  • Note: Data estimated from published figures.

Supp. Fig. 3F: Quantification of Figure 3A Western Blots Mean SD N
Untreated cells H3K9me2/H3 ratio 1 0 3
H3K79me2/H3 ratio 1 0 3
10 mM oct-2-HG treated cells H3K9me2/H3 ratio 3.8 0.5 3
H3K79me2/H3 ratio 8.5 1.5 3
20 mM oct-2-HG treated cells H3K9me2/H3 ratio 5.5 0.3 3
H3K79me2/H3 ratio 17.2 2.4 3
20 mM oct-2-HG + 5 mM oct-α-KG treated cells H3K9me2/H3 ratio 0.6 0.3 3
H3K79me2/H3 ratio 0.9 0.3 3
Test family
  • Due to a lack of raw original data, we are unable to perform power calculations using a MANOVA. We are determining sample size calculations using a two-way ANOVA.

  • Two-way ANOVA followed by Bonferroni corrected comparisons.

Power calculations
ANOVA calculations; α = 0.05
F(1,16) histone Partial η2 Effect size f A priori power Total sample size
235.0200 0.936260 3.83259 99.9%* 10*
*

With 3 samples per group (12 total), achieved power is 99.9%.

Corrected t-tests sample size calculations; α = 0.0083
Group 1 Group 2 Effect size d Power Sample size per group
H3K9me2 Vehicle treated cells 10 mM Oct-2-HG treated cells 11.05934 99.9% 3
H3K79me2 8.92288 99.9% 3
H3K9me2 Vehicle treated cells 20 mM Oct-2-HG treated cells 23.55408 99.9% 3
H3K79me2 14.24069 99.9% 3
H3K9me2 20 mM Oct-2-HG treated cells 20 mM Oct-2-HG + 5 mM oct-α-KG treated cells 28.82353 99.9% 3
H3K79me2 16.46129 99.9% 3
Test family
  • This is an additional analysis to allow a direct comparison with the original study.

  • Bonferroni corrected one-sample t-tests compared to 1 (vehicle treated cells).

Power calculations
  • Calculations were performed with G*Power software, version 3.1.7 (Faul et al., 2007).

Bonferroni corrected t-tests; α = 0.0083
Group Constant Effect size d A Priori power Sample size per group
H3K9me2 10 mM Oct-2-HG treated cells 1 9.65517 90.3% 3
H3K79me2 8.62069 84.4% 3
H3K9me2 20 mM Oct-2-HG treated cells 1 26.47059 99.9% 3
H3K79me2 11.65468 o 96.6% 3

Protocol 3

Summary of original data
  • Note: Data estimated from published figure.

Supp. Figure 3J: quantification of Western blot band intensities from Figure 3D normalized to vector control Mean SD N
With vector + vehicle
H3K4me1/H3 ratio 100 Unspecified 3
H3K4me3/H3 ratio 100 unspecified 3
H3K9me3/H3 ratio 100 unspecified 3
H3K27me2/H3 ratio 100 unspecified 3
H3K79me2/H3 ratio 100 unspecified 3
With IDH1R132H + vehicle
H3K4me1/H3 ratio 209 36 3
H3K4me3/H3 ratio 466 64 3
H3K9me3/H3 ratio 283 56 3
H3K27me2/H3 ratio 232 24 3
H3K79me2/H3 ratio 267 47 3
With IDH1R132H and oct-α-KG
H3K4me1/H3 ratio 105 16 3
H3K4me3/H3 ratio 274 25 3
H3K9me3/H3 ratio 126 21 3
H3K27me2/H3 ratio 99 9 3
H3K79me2/H3 ratio 130 20 3
Test family
  • Due to a lack of raw original data, we are unable to perform power calculations using a MANOVA. We are determining sample size calculations using a two-way ANOVA.

  • Two-way ANOVA followed by Bonferroni corrected comparisons.

Power calculations
ANOVA calculations; α = 0.05
F(1,20) cell treatments Partial η2 effect size f Power Total Sample size
119.5629 0.85670 2.44502 97.1%* 12*
*

With 6 samples per group (for a total of 60 samples), the power achieved is 99.9%.

Corrected t-test sample size calculations; α = 0.005
Group 1 Group 2 Histone Effect size d Power Sample size per group
IDH1R132H + vehicle IDH1R132H + oct-α-KG H3K4me1/H3 ratio 3.73338 94.2%* 5*
H3K4me3/H3 ratio 3.95184 82.1% 4
H3K9me3/H3 ratio 3.71240 93.9% 5
H3K27me2/H3 ratio 7.33811 95.0%§ 3§
H3K79me2/H3 ratio 3.79314 94.9%# 5#
*

With a sample size of 6 per group, the achieved power is 98.9%.

With a sample size of 6 per group, the achieved power is 99.5%.

With a sample size of 6 per group, the achieved power is 98.8%.

§

With a sample size of 6 per group, the achieved power is 99.9%.

#

With a sample size of 6 per group, the achieved power is 99.1%.

Test family
  • Outside the ANOVA framework

  • Bonferroni corrected one-sample t-tests compared to 1 (vector + vehicle).

Power calculations
  • Calculations were performed with G*Power software, version 3.1.7 (Faul et al., 2007).

Corrected t-test sample size calculations; α = 0.005
Group 1 Constant Histone Effect size d Power Sample size per group
IDH1R132H + vehicle 100 H3K4me1/H3 ratio 3.02778 94.2% 6
H3K4me3/H3 ratio 5.71875 92.2%* 4*
H3K9me3/H3 ratio 3.26786 82.9% 5
H3K27me2/H3 ratio 5.50000 90.2% 4
H3K79me2/H3 ratio 3.55319 88.8%§ 5§
*

With a sample size of 6 per group, the achieved power is 99.9%.

With a sample size of 6 per group, the achieved power is 96.9%.

With a sample size of 6 per group, the achieved power is 99.9%.

§

With a sample size of 6 per group, the achieved power is 98.7%.

Protocol 4

Summary of original data
  • ○ Note: Values estimated from published figure.

Figure 7B: Relative 5hmC intensity Mean SD N
50 ng Genomic DNA
Vector 0 0.01 3
TET2-CD 1 0 3
TET2-CM 0 0.01 3
TET2-CD + IDH1 2.5 0.3 3
TET2-CD + IDH1R132H 0.29 0.1 3
TET2-CD + IDH2 2.6 0.11 3
TET2-CD + IDH2R40Q 0.31 0.07 3
TET2-CD + IDH2R172K 0.31 0.09 3
Test family
  • Bonferroni corrected one-sample t-tests compared to 1 (TET2-CD).

Power calculations
  • Power calculations were performed using G*Power software, version 3.1.7 (Faul et al., 2007).

Corrected t-test sample size calculations; α = 0.01
Group 1: TET2 + Constant Effect size d Power Sample size per group
IDH1 1 5.00000 95.9% 4
IDH1R132H 1 7.10000 99.9% 4
IDH2 1 14.54545 99.8%* 3*
IDH2R140Q 1 9.85714 94.6% 3
IDH2R172K 1 7.66667 82.9% 3
*

With a sample size of 4 per group, the achieved power is 99.9%.

With a sample size of 4 per group, the achieved power is 99.9%.

With a sample size of 4 per group, the achieved power is 99.9%.

Protocol 5

Summary of original data
  • Note: Data estimated from published figures.

Figure 8A: TLC blot intensities Mean
TET2 + vehicle 1
TET2 + 10 mM D-2-HG 0.67
TET2 + 25 mM D-2-HG 0.45
TET2 + 50 mM D-2-HG 0.17
TET2 + 10 mM L-2-HG 0.05
TET2 + 25 mM L-2-HG 0.03
TET2 + 50 mM L-2-HG 0.03
Test family
  • One way ANOVA followed by Bonferroni corrected comparisons.

  • Outside the ANOVA framework

    • ○ Bonferroni corrected one-sample t-tests compared to 1 (TET2 + vehicle).

Power calculations
  • Because the original data presented does not have variance (s.e.m. or s.d.), we have performed power calculations using several different levels of calculated variance and an assumed number of replicates to determine a suitable number of replications to perform.

  • Calculations were performed with R software, version 3.1.2 (R Core Team, 2014) and G*Power software, version 3.1.7 (Faul et al., 2007).

Calculated variances and assumed N
Figure 8A: dot blot intensities Mean N 2% 15% 28% 40%
TET2 + vehicle 1 3 n/a* n/a* n/a* n/a*
TET2 + 10 mM D-2-HG 0.67 3 0.0134 0.1005 0.1876 0.268
TET2 + 25 mM D-2-HG 0.45 3 0.009 0.0675 0.126 0.18
TET2 + 50 mM D-2-HG 0.17 3 0.0034 0.0255 0.0476 0.068
TET2 + 10 mM L-2-HG 0.05 3 0.001 0.0075 0.014 0.02
TET2 + 25 mM L-2-HG 0.03 3 0.0006 0.0045 0.0084 0.012
TET2 + 50 mM L-2-HG 0.03 3 0.0006 0.0045 0.0084 0.012
*

Because each replicate will be normalized to TET2 + vehicle this will not have a variance associated with it. And thus the TET2 + vehicle is also not include in the ANOVA calculation.

2% variance

ANOVA calculations; α = 0.05
F(2,12) interaction Partial η2 Effect size f Power Total sample size
1910.6 0.99687 17.8434 98.2%* 9*
*

With 12 total samples, the power achieved is 99.9%.

Corrected t-test sample size calculations; α = 0.01
Group 1 Group 2 Effect size d Power Sample size per group
10 mM D-2-HG 10 mM L-2-HG 65.25231 99.9% 2
50 mM D-2-HG 50 mM L-2-HG 57.34623 99.9% 2
10 mM D-2-HG 50 mM D-2-HG 51.14839 99.9% 2
Corrected t-test sample size calculations; α = 0.01
Group 1: Constant Effect size d Power Sample size per group
10 mM D-2-HG 1 24.62687 97.7% 2
50 mM D-2-HG 1 244.11765 99.9% 2

15% variance

ANOVA calculations; α = 0.05
F(2,12) interaction Partial η2 Effect size f Power Total sample size
2.37930 0.84987 2.37930 93.8% 12*
*

With 12 total samples, the power achieved is 99.9%.

Corrected t-test sample size calculations; α = 0.01
Group 1 Group 2 Effect size d Power Sample size per group
10 mM D-2-HG 10 mM L-2-HG 8.70031 99.9% 3
50 mM D-2-HG 50 mM L-2-HG 7.64616 99.4% 3
10 mM D-2-HG 50 mM D-2-HG 6.81978 97.9% 3
Corrected t-test sample size calculations; α = 0.01
Group 1: Constant Effect size d Power Sample size per group
10 mM D-2-HG 1 3.28358 87.2% 6
50 mM D-2-HG 1 32.54902 99.5% 3

28% variance

ANOVA calculations; α = 0.05
F(2,12) interaction Partial η2 Effect size f Power Total sample size
9.7548 0.61916 1.27507 86.1% 12
Corrected t-test sample size calculations; α = 0.01
Group 1 Group 2 Effect size d Power Sample size per group
10 mM D-2-HG 10 mM L-2-HG 4.66088 97.9% 4
50 mM D-2-HG 50 mM L-2-HG 4.09616 93.6% 4
10 mM D-2-HG 50 mM D-2-HG 3.65346 86.6% 4
Corrected t-test sample size calculations; α = 0.01
Group 1: Constant Effect size d Power Sample size per group
10 mM D-2-HG 1 1.75906 80.3% 14
50 mM D-2-HG 1 17.43697 94.0% 4

40% variance

ANOVA calculations; α = 0.05
F(2,12) interaction Partial η2 Effect size f Power Total sample size
4.7765 0.44323 0.892237 82.2% 17*
*

With 18 total samples, the power achieved is 85.3%.

Corrected t-test sample size calculations; α = 0.01
Group 1 Group 2 Effect size d Power Sample size per group
10 mM D-2-HG 10 mM L-2-HG 3.26262 92.8% 5
50 mM D-2-HG 50 mM L-2-HG 2.86731 83.9% 5
10 mM D-2-HG 50 mM D-2-HG 2.55742 86.3% 6
Corrected t-test sample size calculations; α = 0.01
Group 1: Constant Effect size d Power Sample size per group
10 mM D-2-HG 1 1.23134 80.8% 27
50 mM D-2-HG 1 12.20588 84.6% 5

In order to produce quantitative replication data, we will run the experiment six times. Each time we will quantify band intensity. We will determine the standard deviation of band intensity across the biological replicates and combine this with the reported value from the original study to simulate the original effect size. We will use this simulated effect size to determine the number of replicates necessary to reach a power of at least 80%. We will then perform additional replicates, if required, to ensure that the experiment has more than 80% power to detect the original effect.

Acknowledgements

The Reproducibility Project: Cancer Biology core team would like to thank the original authors, in particular Dr. Yue Xiong, for generously sharing reagents to ensure the fidelity and quality of this replication attempt. We would also like to thanks the following companies for generously donating reagents to the Reproducibility Project: Cancer Biology; American Tissue Culture Collection (ATCC), Applied Biological Materials, BioLegend, Charles River Laboratories, Corning Incorporated, DDC Medical, EMD Millipore, Harlan Laboratories, LI-COR Biosciences, Mirus Bio, Novus Biologicals, Sigma–Aldrich, and System Biosciences (SBI).

Funding Statement

The Reproducibility Project: Cancer Biology is funded by the Laura and John Arnold Foundation, provided to the Center for Open Science in collaboration with Science Exchange. The funder had no role in study design or the decision to submit the work for publication.

Footnotes

Xu U, Yang H, Liu Y, Yang Y, Wang P, Kim SH, Ito S, Yang C, Wang P, Xiao MT, Liu LX, Jiang WQ, Liu J, Zhang JY, Bin W, Frye S, Zhang Y, Xu YH, Lei QY, Guan KL, Zhao SM, Xiong Y. 2011. Oncometabolite 2-hydroxyglutarate is a competitive inhibitor of α-ketoglutarate-dependent dioxygenases. Cancer Cell 4:17–30. doi: 10.1016/j.ccr.2010.12.014.

Contributor Information

Irwin Davidson, Institut de Génétique et de Biologie Moléculaire et Cellulaire, France.

Reproducibility Project: Cancer Biology:

Elizabeth Iorns, William Gunn, Fraser Tan, Joelle Lomax, and Timothy Errington

Funding Information

This paper was supported by the following grant:

  • Laura and John Arnold Foundation to .

Additional information

Competing interests

RP:CB: We disclose that EI, FT, and JL are employed by and hold shares in Science Exchange Inc. The experiments presented in this manuscript will be conducted by BE at the Proteomics and Mass Spectrometry Facility, which is a Science Exchange lab.

The other authors declare that no competing interests exist.

Author contributions

BE, Drafting or revising the article.

EG, Drafting or revising the article.

RP:CB, Conception and design, Drafting or revising the article.

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eLife. 2015 Jul 31;4:e07420. doi: 10.7554/eLife.07420.002

Decision letter

Editor: Irwin Davidson1

eLife posts the editorial decision letter and author response on a selection of the published articles (subject to the approval of the authors). An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent (see review process). Similarly, the author response typically shows only responses to the major concerns raised by the reviewers.

Thank you for submitting your work entitled “Registered report: Oncometabolite 2-Hydroxyglutarate Is a Competitive Inhibitor of α-Ketoglutarate-Dependent Dioxygenases” for peer review at eLife. Your submission has been favorably evaluated by Michael Marletta (Senior editor), Irwin Davidson (Reviewing editor), and four reviewers.

The reviewers have discussed the reviews with one another and the Reviewing editor has drafted this decision to help you prepare a revised submission. The reviewers of this paper have raised several issues and we would ask you to specifically take into account the comments concerning the statistical analyses.

Summary:

The article outlines the detailed protocol to reproduce a report published in Cancer Cell linking mutations in IDH1/2 to cellular levels of Ketoglutarate and changes in histone and DNA methylation. This initial work had a major impact on linking metabolites to chromatin, but also raised a number of questions that justify a rigorous replication.

Overall, the proposed study covers the major aspects with the required detail and rigour.

Specific points to address are:

1) The referees suggest that the authors consider using mass spectrometry to measure 5hmC in addition to immune dot-blot. Mass spectrometry is a more quantitative measure and while it would go beyond replicating the published findings it might give a clearer answer.

2) In protocol 1 a 2-way ANOVA is proposed, however as 2 quantitative variables are measured and there is only one qualitative factor (with three possible values) influencing these measures, an MANOVA would be more suited.

3) There may be confusion between groups and variables in setting the degrees of freedom for ANOVA analyses. In protocol 1, how was (2, 6) obtained? The same question applies to protocols 3, 4 and 5.

4) In addition to t-tests for the comparison of means where both variances are equal, F-tests should be added when variances are significantly different.

5) Referees raised concerns about null variances that appear in the power calculation tables. Although these values are not always available, variance values can change the conclusion of the tests. When variances are not available, preliminary experiments in order to estimate them are proposed. More generally, variance values used in this paper are estimated from published figures using a low number of replicates, so they are not robust. A way to increase robustness would be to increase measured values by a pre-determined factor and then relax the expected power if too many replicates are required.

Also, non-rounded computed sample sizes are requested to have an idea of how close we are to the theoretical value after rounding.

6) For protocol 2, in subsection “Confirmatory analysis plan”, a MANOVA is proposed, whereas a one-way ANOVA is suggested for the same protocol in subsection “Test family”. Please correct this inconsistency.

7) Protocol 5: a mean across several groups is compared with the mean of a single group. This should not be done with a simple t-test to take into account the fact that the number of measures is different in the two groups being compared.

eLife. 2015 Jul 31;4:e07420. doi: 10.7554/eLife.07420.003

Author response


1) The referees suggest that the authors consider using mass spectrometry to measure 5hmC in addition to immune dot-blot. Mass spectrometry is a more quantitative measure and while it would go beyond replicating the published findings it might give a clearer answer.

We agree mass spectrometry analysis would be a more quantitative approach to measure 5hmC levels, however feel it is beyond the scope of this project, which is to perform a direct replication of the original experiment(s). Aspects of an experiment not included in the original study are occasionally added to ensure the quality of the research, but by no means is a requirement of this project; rather, it is an extension of the original work. We know that the exclusion of certain experiments limits the scope of what can be analyzed by the project, but we are attempting to identify a balance of breadth of sampling for general inference with sensible investment of resources on replication projects to determine to what extent the included experiments are reproducible.

2) In protocol 1 a 2-way ANOVA is proposed, however as 2 quantitative variables are measured and there is only one qualitative factor (with three possible values) influencing these measures, an MANOVA would be more suited.

We agree and have included this in the confirmatory analysis section. However, as we do not have the raw data needed to perform a power calculation for the MANOVA test, we performed it with a 2-way ANOVA to estimate the needed sample size and adjusted the alpha error for the planned contrasts that will be performed to ensure the sample size is sufficient.

3) There may be confusion between groups and variables in setting the degrees of freedom for ANOVA analyses. In protocol 1, how was (2, 6) obtained? The same question applies to protocols 3, 4 and 5.

Thank you for catching these inconsistencies, we have checked and adjusted each protocol in regards to degrees of freedom. Some of the analysis sections have changed to address other questions below, but a link to all scripts has been provided below and in the revised manuscript.

4) In addition to t-tests for the comparison of means where both variances are equal, F-tests should be added when variances are significantly different.

We have added a note in the analysis section that at the time of analysis, we will assess the normality and homoscedasticity of the data. If necessary, we will perform the appropriate transformation in order to proceed with the proposed statistical analysis. We will note any changes or transformations made. We have updated the manuscript to address this point.

5) Referees raised concerns about null variances that appear in the power calculation tables. Although these values are not always available, variance values can change the conclusion of the tests. When variances are not available, preliminary experiments in order to estimate them are proposed. More generally, variance values used in this paper are estimated from published figures using a low number of replicates, so they are not robust. A way to increase robustness would be to increase measured values by a pre-determined factor and then relax the expected power if too many replicates are required.

Also, non-rounded computed sample sizes are requested to have an idea of how close we are to the theoretical value after rounding.

We agree about the concern about null variances and have reanalyzed the proposed analysis plans and power calculations to reflect this. In many cases the reason for the null variance was due to normalization to a common factor within each replicate – thus making the variance zero on purpose. In some cases we will need to repeat this as well (protocol 3, 4, and 5). In other cases (protocol 1 and 2) we used the other variances that were reported as an estimate for the null variances. And where possible (protocol 2) we included additional analysis to allow a direct comparison to the original analysis.

Full details of all power calculations are available through the study’s page on the Open Science Framework (https://osf.io/gnsti/?view_only=f3a48d5a355f429fa2264ee7c17e9705). Unfortunately, the program we use to calculate sample sizes, G*Power, only returns whole integers for recommended sample sizes.

6) For protocol 2, in subsection “Confirmatory analysis plan”, a MANOVA is proposed, whereas a one-way ANOVA is suggested for the same protocol in subsection “Test family”. Please correct this inconsistency.

The Confirmatory analysis plan is in reference to the proposed statistical analyses of the replication data. However, as we do not have the raw data needed to perform a power calculation for the MANOVA test, we performed it with a 2-way ANOVA to estimate the needed sample size and adjusted the alpha error for the planned contrasts that will be performed to ensure the sample size is sufficient.

7) Protocol 5: a mean across several groups is compared with the mean of a single group. This should not be done with a simple t-test to take into account the fact that the number of measures is different in the two groups being compared.

We had originally intended to perform a weighted planned contrast by comparing several groups to a single group – and agree an F test is properly suited. However, in the revised manuscript we are including multiple independent t-tests and one-sample t-tests (due to the necessary normalization described in point 5 above) to more thoroughly analyze the data as originally reported and interpreted.


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