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. Author manuscript; available in PMC: 2021 Jul 6.
Published in final edited form as: Methods Mol Biol. 2017;1507:113–127. doi: 10.1007/978-1-4939-6518-2_9

eIF3 Regulation of Protein Synthesis, Tumorigenesis, and Therapeutic Response

Ji-Ye Yin 1, Zizheng Dong 2, Jian-Ting Zhang 2
PMCID: PMC8259889  NIHMSID: NIHMS1713362  PMID: 27832536

Abstract

Translation initiation is the rate-limiting step of protein synthesis and highly regulated. Eukaryotic initiation factor 3 (eIF3) is the largest and most complex initiation factor consisting of 13 putative subunits. A growing number of studies suggest that eIF3 and its subunits may represent a new group of proto- oncogenes and associates with prognosis. They regulate translation of a subset of mRNAs involved in many cellular processes including proliferation, apoptosis, DNA repair, and cell cycle. Therefore, unveiling the mechanisms of eIF3 action in tumorigenesis may help identify attractive targets for cancer therapy. Here, we describe a series of methods used in the study of eIF3 function in regulating protein synthesis, tumorigenesis, and cellular response to therapeutic treatments.

Keywords: Eukaryotic initiation factor 3 (eIF3), Translational control, Protein synthesis, Tumorigenesis, Therapeutic response

1. Introduction

Gene expression is regulated primarily at levels of transcription and translation. Translation is a process of transferring genetic information from mRNA to protein, and, thus, its deregulation results in abnormal gene expression. Aberrant protein synthesis and dysregulation of mRNA translation have been associated with diseased state such as cancer [1–5]. In eukaryotes, mRNA translation is a complicated process consisting of three major steps: initiation, elongation, and termination [6]. Translation initiation is the rate-limiting step of protein synthesis and therefore highly regulated. Eukaryotic initiation factors (eIFs) are proteins that play important roles in the initiation step.

Among all human eIFs, eIF3 is the largest and most complex one consisting of 13 putative subunits, which are named as eIF3a to eIF3m. A growing number of studies suggest that eIF3 subunits may associate with tumorigenesis and therapeutic response through regulating protein synthesis [3, 7, 8]. Here, we describe a series of techniques and methods used in the study of eIF3 in regulating protein synthesis, tumorigenesis, and therapeutic response.

2. Materials

2.1. Reagents

  1. EDTA.

  2. Goat and fetal bovine serum.

  3. H2O2.

  4. Phosphate-Buffered Saline (PBS).

  5. Bovine serum albumin (BSA).

  6. Propidium iodide (25 μg/ml).

  7. AMV reverse transcriptase.

  8. Reverse Transcription System Kit (Promega, Madison, WI, USA).

  9. Fluorescent SYBR Green dyes.

  10. Culture media supplemented with 10 % serum.

  11. Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

  12. Dimethyl sulfoxide (DMSO).

  13. Annexin V-FITC Apoptosis Detection Kit I (BD Pharmingen, San Jose, CA, USA).

  14. DAPI or Hoechst-33342 dye.

  15. [35S] methionine.

  16. Trichloroacetic acid (TCA).

  17. Cycloheximide (CHX).

  18. RNeasy Mini Kit (Qiagen, Hilden, Germany).

  19. T7 or SP6 RNA polymerase.

  20. rNTP.

  21. Dithiothreitol (DTT).

  22. RNasin.

  23. m7GpppG.

  24. DNase I.

  25. Rabbit reticulocyte lysate (RRL) system (Promega, Madison, WI, USA).

  26. Opti-MEM I (Invitrogen, Carlsbad, CA, USA).

  27. Lipofectin (Invitrogen, Carlsbad, CA, USA).

  28. Passive Lysis Buffer (Promega, Madison, WI, USA).

  29. Luciferase Reporter Assay Kit (Promega, Madison, WI, USA).

  30. α[32P]-UTP.

  31. RNase mixture (4 μg/μl RNase A and 5 U/μl RNase T1).

  32. Biotin-11-CTP.

  33. Streptavidin MagneSphere Paramagnetic Particles (Promega, Madison, WI, USA).

  34. Phenol/chloroform/isoamyl alcohol.

  35. Ammonium acetate.

  36. Lithium chloride.

  37. Glycogen.

  38. 100% ethanol.

  39. RNase-free water.

2.2. Buffers and Solutions

  1. Lysis buffer 1 [50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 % Triton X-100, 1 % sodium deoxycholate, 0.1 % SDS, 1 % NP-40, 1 mM sodium orthovanadate, 1 mM EDTA, 1 mM sodium fluoride, 100 mg/ml phenylmethylsulfonyl fluoride (PMSF), 100 mg/ml DTT].

  2. 1× binding buffer 1 [10 mM Hepes/NaOH (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2].

  3. Polysome extraction buffer [10 mM Tris–HCl (pH 7.4), 150 mM NaCl, 5 mM MgCl2, 1 % Triton X-100, 40 mM DTT, 0.1 mg/ml CHX, and 1 mg/ml heparin].

  4. Binding buffer 2 [20 mM Hepes (pH 7.9), 100 mM KCl, 10 % glycerol, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, and 5 mM MgCl2].

  5. Wash buffer 1 (10 mM (Hepes), 20 mM KCl, 1 mM MgCl2, 1 mM DTT, and 1 mM PMSF).

  6. Lysis buffer 2 (10 mM Hepes, pH 7.0, 100 mM KCl, 5 mM MgCl2, 0.5 % NP-40, 1 mM DTT, 100 units/ml RNase inhibitor, protease inhibitor cocktail).

  7. Wash buffer 2 (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 0.05 % NP-40).

  8. Immunoprecipitation buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 0.05 % NP-40, 20 mM EDTA, pH 8.0, 1 mM DTT, 100 units/ml RNase inhibitor).

  9. Proteinase K solution (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 0.05 % NP-40, 1 % SDS, 1.2 mg/ml proteinase K).

  10. Crystal violet staining solution (0.5 % crystal violet in 20 % methanol).

  11. 0.6 % and 0.3 % agar solution in cell culture medium.

  12. 10 % formalin buffer.

  13. 5 mg/ml thiazolyl blue tetrazolium bromide solution in 1× PBS.

2.3. Plasmids and siRNAs

  1. pCβA vector control.

  2. pCβA-eIF3 subunit.

  3. eIF3 subunit siRNAs.

  4. Scrambled control siRNAs.

3. Methods

3.1. Detection of EIF3 Expression and Localization

Expression of various eIF3 subunits has been found to be altered in various human cancers [7], and it has also been found that eIF3a may localize in different subcellular compartments [9, 10]. Thus, to investigate the expression of eIF3 subunits and their subcellular localization, it is necessary to detect these proteins effectively in either cell line models or in clinical samples. For studies involving manipulation of eIF3 expression using ectopic overexpression or RNA interference knockdowns, it is also necessary to effectively detect these proteins using different methods.

3.1.1. Western Blot

  1. Cells are lysed in lysis buffer 1 [50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 % Triton X-100, 1 % sodium deoxycholate, 0.1 % SDS, 1 % NP-40, 1 mM sodium orthovanadate, 1 mM EDTA, 1 mM sodium fluoride, 100 mg/ml phenylmethylsulfonyl fluoride (PMSF), 100 mg/ml dithiothreitol (DTT)] at 4 °C for 30 min.

  2. Protein concentration of the lysate is determined using the Bradford method.

  3. Then, protein samples are separated by 10 % sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes.

  4. The membranes are then blocked with 5 % nonfat milk and incubated with eIF3 subunit antibodies overnight followed by washing and incubation with horseradish peroxidase-conjugated secondary antibodies.

  5. The reaction is detected using ECL reagents, and the signals are captured by X-ray films or by imaging systems.

3.1.2. Immunohisto chemistry (IHC)

  1. To conduct IHC staining, serial 4-mm thick sections are cut from tissue blocks and mounted on slides. The slide containing maximum amount of tumors is selected for each case, and one representative slide from each case is used.

  2. Sample sections on slides are first baked at 60 °C for 30 min followed by incubation in xylene for 2×10 min and rehydration through graded ethanol to distilled water.

  3. Antigen retrieval is done by heating samples in 1 mmol/L EDTA (pH 8.0) for 20 min.

  4. Nonspecific staining is blocked by 10 % goat serum in PBS buffer for 20 min at room temperature, and endogenous peroxidase activity is quenched by incubation in 3 % H2O2 for 10 min.

  5. Slides are then incubated with eIF3 subunit antibody/antibodies or PBS control at 4 °C overnight followed by incubation with biotinylated secondary antibody and peroxidase-conjugated streptavidin.

  6. The staining is then visualized by using 3,3′-diaminobenzidine tetrahydrochloride substrate, and all samples are counterstained with hematoxylin and eosin (H&E) before viewing with an inverted microscope (see Note 1).

3.1.3. Immunofluore scence (IF)

  1. For immunofluorescence staining, cells are seeded on a glass coverslip in a six-well plate and allowed to grow until near confluence.

  2. The cells on cover glass are washed three times with ice-cold phosphate-buffered saline and fixed with acetone/methanol (1:1) at room temperature for 10 min and incubated at 4 °C for 30 min with blocking solution (1 % BSA in PBS).

  3. The cells are then probed with eIF3 antibodies for 1 h at 4 °C followed by incubation with secondary antibody conjugated with fluorescein isothiocyanate (FITC) at 4 °C for another 1 h.

  4. After washing three times with blocking solution, cell nuclei are counterstained with propidium iodide (25 μg/ml) for 10 min ( see Note 2).

  5. The coverslips are then mounted on the slides and viewed with a confocal microscope.

3.1.4. Real-Time Reverse Transcription-Polymerase Chain Reaction (RT-PCR)

  1. Firstly, total RNA is isolated using RNeasy Mini Kit (Qiagen, Hilden, Germany).

  2. 1 μg of the total RNAs is reverse-transcribed with 0.5 μg random primers and 15 unit AMV reverse transcriptase in a total volume of 20 μl at 42 °C for 1 h using Reverse Transcription System Kit (Promega, Madison, WI, USA).

  3. The real-time PCR is then carried out in a real-time PCR system with fluorescent SYBR Green dyes.

  4. The cycle threshold value (Ct) is defined as the PCR cycle number at which the reporter fluorescence crosses the threshold. The Ct of each product is determined and normalized against that of the internal control (β-actin or GAPDH).

3.2. EIF3-Induced Tumorigenic Assays

A large number of studies showed that some eIF3 subunits may play important roles in tumorigenesis, including eIF3a, eIF3b, eIF3c, eIF3h, and eIF3i, using ectopic overexpression and siRNA knockdown [11–22] and assays detecting the effect of eIF3 on cell growth, apoptosis, malignant transformation, and tumor formation.

3.2.1. Ectopic Overexpression and Knockdown of eIF3

  1. Cells are seeded in a 6-well plate and maintained in culture media supplemented with 10 % serum (see Note 3).

  2. It is followed by transfection with 4 μg pCβA vector control and eIF3 subunit-expressing constructs for overexpression or 50 nmol/L eIF3 subunit and scrambled control siRNAs for knockdown using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).

  3. Following incubation for 48 h post-transfection, cells can be harvested for further analysis.

  4. To establish stable clones with eIF3 subunit overexpression, the transiently transfected cells are collected 24 h post-transfection and replated in 100-mm dishes followed by selection with 0.6–1.0 mg/mL G418 for 2 weeks.

  5. The G418-resistant clones are further propagated for testing eIF3 expression using real-time PCR and Western blot analyses as described above. The positive stable clones are then maintained in the presence of 0.2 mg/mL G418 for further analysis.

3.2.2. Colony Formation and Anchorage-Independent Growth Assay

  1. Colony formation assay is performed by seeding 100 cells per well in six-well plates and cultured for 10–14 days with medium changed every 2–3 days.

  2. At the end of the assay, cell colonies are stained with crystal violet (0.5 % crystal violet in 20 % methanol) for 20 min and washed thoroughly with water. The visible colonies are counted manually.

  3. For anchorage-independent growth assay, cells are suspended at a density of 2.5 × 103 cells/ml in 0.3 % agar solution in cell culture medium.

  4. 1 ml of this suspension is overlaid on top of a 0.6 % agar layer made in cell culture medium in a six-well plate.

  5. Cells are then cultured for 14–25 days with fresh medium changed every 2–3 days.

  6. At the end of assays, the cells are stained as described above and visible colonies are counted manually.

3.2.3. Xenograft Tumor Formation Assay

  1. Xenograft tumor formation in immune deficient mice (see Note 4) is an essential assay to investigate the tumorigenesis function of eIF3 genes.

  2. Approximately 1 × 107 cells are injected subcutaneously into 7-week-old nonobese diabetic/severe combined immunodeficient (NOD/SCID) or nude mice (one injection/mouse).

  3. Tumor growth is measured by a caliper twice a week for a total of 4–9 weeks. The tumor volume is calculated from two perpendicular diameters using the formula: volume = (length/2) × (width2).

  4. To confirm the xenograft tumor pathology, tumor tissues are removed, measured, and fixed in 10 % formalin buffer.

  5. After staining with H&E, samples are subjected to standard histology and pathology analyses.

3.3. Therapeutic Response

In addition to the potential role of eIF3 in tumorigenesis, eIF3 has also been found to associate with prognosis [14, 23–26] possibly by regulating cellular response to anticancer drugs [14, 27]. Thus, assays to analyze cellular response to different anticancer drugs are important for studying the role of eIF3 in cell survival against anticancer drugs and in drug-induced apoptosis.

3.3.1. Survival Methyl Thiazolyl Tetrazolium (MTT) Assay

  1. MTT assay is the easiest and one of the most commonly used approaches for studying cell survival.

  2. Cells are seeded in 96-well plates at a density of 2000 cells/ well and cultured for 24 h before treating the cells with anticancer drugs at different concentrations for 3 days.

  3. The culture medium is then removed, and thiazolyl blue tetrazolium bromide is added to a final concentration of 0.5 mg/ml followed by incubation for 4 h at 37 °C.

  4. The formazan is then solubilized by adding 150 μl/well dimethyl sulfoxide (DMSO) and OD570nm is measured in a microplate reader.

  5. The half of maximal inhibitory concentration (IC50) values can be obtained from the dose–response curves.

  6. Alternatively, colony formation assay as described above can be used to investigate survival following drug treatments.

3.3.2. Apoptosis Assay

Apoptosis can be detected using several assays, and in the following section, we describe two assays that have been used to study eIF3. Annexin V staining is a most commonly used method to quantitatively measure apoptotic cell populations:

  1. Firstly, cells are seeded in 6-well plate and allowed to attach overnight.

  2. Then, the medium is replaced with fresh one containing anticancer drugs and the cells are cultured for 24 h.

  3. The cells are then washed twice with ice-cold phosphate-buffered saline and resuspended in 1× binding buffer 1 [10 mM Hepes/NaOH (pH 7.4), 140 mM NaCl, 2.5 mM CaCl2] at 1 × 106 cells/ml and incubated with FITC-conjugated Annexin V antibody and propidium iodide counterstain for DNA using Annexin V-FITC Apoptosis Detection Kit I (BD Pharmingen, San Jose, California, USA).

  4. About 10,000 cells in each sample are analyzed using a BD FACSCalibur flow cytometer. Cell apoptosis profiles are analyzed using BD CellQuestPro software.

  5. Another assay is based on the staining of disintegrated nuclei by using DAPI or Hoechst-33342.

  6. Briefly, cells following anticancer drug treatment are harvested and stained with 1 μg/ml DAPI or 1 μg/ml Hoechst-33342 dye.

  7. The stained cells are then mounted onto a polylysine-coated slide by centrifugation and examined under a fluorescent microscope.

  8. A total of 300–400 nuclei from five randomly chosen fields are examined, and the nuclei displaying distinctive apoptosis-associated morphological changes are scored. Apoptosis can be quantified as a fraction of the total number of nuclei examined.

3.4. Protein Synthesis Assays

One mechanism of eIF3 regulation of cell proliferation and response to insults is that they differentially regulate translation of a subset of specific cancer and survival-related mRNAs [13, 28, 29]. Several assays can be used to detect translational regulation.

3.4.1. Metabolic Labeling

  1. [35S]methionine labeling in live cells is widely used to measure total or specific protein synthesis.

  2. Briefly, 1 × 105 cells per well are seeded in a 24-well plate in triplicates and incubated for 24 h followed by washing three times with serum-free and methionine-free medium.

  3. The cells are then incubated with methionine-free media supplemented with 20 μCi/ml [35S]methionine for 30 min, washed for three times, and harvested.

  4. Next, cells are lysed followed by separation on SDS-PAGE and autoradiography analysis for determination of global protein synthesis or immunoprecipitation of specific proteins of interest and then separation on SDS-PAGE and autoradiography analysis of specific proteins.

  5. Alternatively, cell lysates are precipitated with 10 % TCA. The acid-insoluble material is collected on a filter by rapid filtration, and the radioactivity is determined by scintillation counting to quantify global protein synthesis.

3.4.2. Polysome Profiling Analysis

  1. Polysomes are defined as mRNA bound with multiple ribosomes during translation; it represents actively translated mRNAs [30]. Thus, analyzing polysomes using high-throughput methods enables profiling of actively translated mRNAs.

  2. Approximately 5 × 107 cells are incubated at 37 °C prior to the experiment. Cycloheximide (CHX) is firstly added into the medium at a final concentration of 0.1 mg/ml for 10 min at 37 °C.

  3. The medium is then removed and cells are washed twice with cold PBS containing 0.1 mg/ml CHX followed by harvesting cells.

  4. The cells are lysed using 500 μl polysome extraction buffer [10 mM Tris–HCl (pH 7.4), 150 mM NaCl, 5 mM MgCl2, 1 % Triton X-100, 40 mM DTT, 0.1 mg/ml CHX, and 1 mg/ ml heparin].

  5. Extracts are incubated on ice for 15 min with occasional vortexing followed by centrifugation to remove nuclei and debris. 500 μl supernatant is recovered and layered onto 10 ml, 10–50 % linear sucrose gradients in extraction buffer lacking Triton X- 100.

  6. The gradients are centrifuged at 38,000 × ɡ for 3 h at 4 °C.

  7. Then polysome profiles are monitored by collecting fractions and concomitant measurement of the absorbance at 254 nm. Polysomal RNAs are extracted using RNeasy Mini Kit (Qiagen, Hilden, Germany) and subjected to further analysis (see Note 5).

3.4.3. In Vitro Transcription and Translation

  1. In vitro translation in rabbit reticulocyte lysate can be programed using cRNAs, which can also be generated using in vitro transcription [31–34].

  2. To perform in vitro transcription, cDNA templates are linearized by digestion with restriction endonuclease, and cRNA transcripts carrying both 5′-cap and 3′-poly(A) tail are synthesized by incubating 15 units T7 or SP6 RNA polymerase in the presence of 5 μg linearized DNA, 0.5 mM rNTP, 10 mM DTT, 60 units RNasin, and 30 mM m7 GpppG in a final volume of 50 μl at 37 °C for 1.5 h.

  3. After digestion with 3 units DNase I at 37 °C for 15 min, the in vitro cRNA transcripts are then purified using RNeasy Mini Kit (Qiagen, Hilden, Germany).

  4. For in vitro translation, about 50 ng of in vitro capped cRNA transcripts are used to program cell-free translation in rabbit reticulocyte lysate (RRL) system (Promega, Madison, WI, USA) in a final volume of 10 μl containing 3.5 μl RRL in the presence of [35S] methionine and purified eIF3a proteins followed by SDS-PAGE separation and autoradiography analyses.

3.4.4. RNA-Based Luciferase Reporter Assay

  1. RNA-Based Luciferase Reporter Assay can quantitatively measure translational regulation in a convenient way and to study the role of UTRs in translational control.

  2. For this assay, in vitro transcripts with specific UTRs of interests are advised to be used in place of DNA constructs encoding the transcripts.

  3. Briefly, 2 × 105 cells/well are seeded into 6-well plates on the day before transfection. Cells are then washed once with Opti-MEM I (Invitrogen, Carlsbad, CA, USA) with reduced serum medium and incubated with a mixture containing 12.5 μg Lipofectin (Invitrogen, Carlsbad, CA, USA), 1 μg transcripts, and 1 ml Opti-MEM I medium.

  4. At 8 h after transfection, cells are lysed in 500 μl 1× Passive Lysis Buffer (Promega, Madison, WI, USA). The luciferase activities can be determined using Luciferase Reporter Assay Kit (Promega, Madison, WI, USA).

3.5. EIF3-mRNA Binding Assays

eIF3 proteins have been reported to regulate translation of a subset of specific cancer-related mRNAs possibly by directly binding to these mRNAs [11, 29, 31]. Therefore, assays to assess eIF3– nucleic acid interactions are important for understanding the molecular mechanism of eIF3 function in translational regulation.

3.5.1. UV Cross-Linking

  1. UV cross-linking is a powerful method to detect RNA-binding proteins [11, 31]. UV irradiation can trigger the formation of covalent bond between RNA and its binding proteins.

  2. RNA probes are firstly generated using in vitro transcription as described above in the presence of 70 μCi α[32P]-UTP and purified using RNeasy Mini Kit (Qiagen, Hilden, Germany).

  3. The RNA probe is then diluted to 1 × 105 cpm/μl and mixed with 20 μg purified eIF3 proteins or total cell lysate and 30 μg E. coli tRNA in binding buffer 2 [20 mM Hepes (pH 7.9), 100 mM KCl, 10 % glycerol, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, and 5 mM MgCl2] followed by incubation at RT for 30 min and irradiation by UV (254 nm, 5.4 J/cm2).

  4. Following digestion using 10 μl RNase mixture (4 μg/μl RNase A and 5 U/μl RNase T1) at 37 °C for 30 min, the mixture can be separated by SDS-PAGE for autoradiography if purified protein is used.

  5. In the case of cell lysate, the reaction mixture is subjected to immunoprecipitation of the eIF3 protein of interest followed by SDS-PAGE separation and autoradiography.

3.5.2. RNA Electrophoretic Mobility Shift Assay (EMSA)

  1. EMSA is a standard affinity electrophoresis method to detect protein–nucleic acid (DNA or RNA) interactions [11, 35, 36].

  2. For RNA EMSA, RNA probes are firstly generated using in vitro transcription in the presence of α[32P]-UTP and purified by using RNeasy Mini Kit as described above. Then 1–5 μg purified proteins or total cell lysate is mixed with 200 μg/mL of yeast tRNA, in binding buffer [10 mM HEPES (pH 7.9), 50 mM KCl, 10 % glycerol, 0.2 mg/ml BSA, 1 mM DTT, and 0.2 mM PMSF], and 4 × 104 cpm α[32P]-labeled RNA probes.

  3. The mixture is incubated for 30 min at room temperature and unbound probes are digested by 100 units RNase T1 for 15 min at 30 °C. The reaction mixtures are then separated on non-denaturing PAGE. The signal is detected by a utoradiography (see Note 6).

3.5.3. Pulldown Assay Using Biotinylated RNA probe

  1. Pulldown assays using biotinylated RNA probes have an advantage of avoiding the use of radio isotopes [11, 31].

  2. The biotinylated RNA probes are generated using in vitro transcription as described above but in the presence of 0.625 mM Biotin-11-CTP and purified as described above.

  3. Biotinylated RNA probe is incubated with 20 μg purified eIF3 proteins or total cell lysate and 30 μg E. coli tRNA in binding buffer 2 [20 mM Hepes (pH 7.9), 100 mM KCl, 10 % glycerol, 1 mM EDTA, 1 mM DTT, 1 mM PMSF, and 5 mM MgCl2] at RT for 1 h and followed by UV irradiation (254 nm, 5.4 J/cm2).

  4. The RNA–protein complexes are then digested by 10 μl RNase mix (4 μg/μl RNase A and 5 U/μl RNase T1) at 37 °C for 30 min and isolated using 0.5 mg/ml Streptavidin MagneSphere Paramagnetic Particles (Promega, Madison, WI, USA) at room temperature for 30 min, followed by washing for three times with wash buffer 1 (10 mM Hepes, 20 mM KCl, 1 mM MgCl2, 1 mM dithiothreitol, and 1 mM PMSF).

  5. The pulldown materials are then separated by SDS-PAGE and analyzed using Western blot probed by antibodies specific to the eIF3 proteins of interest.

3.5.4. RNA Immunoprecipitation

  1. The above methods are directed for studies using in vitro transcribed RNA probes. To investigate interaction between endogenous proteins and RNAs, RNA immunoprecipitation can be used.

  2. Firstly, cells are lysed in lysis buffer 2 (10 mM Hepes, pH 7.0, 100 mM KCl, 5 mM MgCl2, 0.5 % NP-40, 1 mM DTT, 100 units/ml RNase inhibitor, protease inhibitor cocktail) by pipetting up and down 7–10 times followed by incubation on ice for 5 min and then freezing at −80 °C overnight.

  3. Next day, protein A-conjugated magnetic beads are washed with wash buffer 2 (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 0.05 % NP-40) for one time and then resuspended in 100 μl wash buffer containing 5 μg eIF3 antibody followed by incubation for 30 min at room temperature, washing for three times, and resuspended in 900 μl immunoprecipitation buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 0.05 % NP-40, 20 mM EDTA, pH 8.0, 1 mM DTT, 100 units/ml RNase inhibitor).

  4. The cell lysate is thawed quickly and cleared by centrifugation at 15,000 × ɡ for 10 min at 4 °C. Aliquot 10 μl of the cell lysate and mark it as input and store it at −80 °C. Mix 100 μl cell lysate to the bead suspension above and incubate at 4 °C for 4–6 h or overnight with agitation. At the end of incubation, the beads are collected and washed with the wash buffer for 6 times. Finally, the beads are resuspended in 150 μl proteinase K solution (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM MgCl2, 0.05 % NP-40, 1 % SDS, 1.2 mg/ml proteinase K), while the cell lysate input is thawed and mixed with 140 μl proteinase K solution, followed by incubation at 55 °C for 45 min and mixing with 250 μl wash buffer to make the final volume to 400 μl.

  5. The mixtures are then extracted with phenol/chloroform/isoamyl alcohol. The RNA is precipitated by adding 1/6 volume 5 M ammonium acetate, 1/20 volume 7.5 M lithium chloride, 1/60 volume 5 mg/ml glycogen, and 2.5 volume ethanol and storing at −80 °C overnight. The RNAs are recovered and dissolved in 20 μl RNase-free water.

  6. Take 6 RNA μL samples to do reverse transcription to synthesize cDNA and analyze by quantitative PCR.

Acknowledgment

This work was supported in part by the National Natural Science Foundation of China Grants 81573463, Hunan Provincial Natural Science Foundation of China Grant 2015JJ1024, and National Institutes of Health Grant R01 CA140582.

Footnotes

1.

Quantification of IHC staining is difficult but important to be performed. All IHC staining of tissue sections should be evaluated independently by at least two pathologists. Microscopic fields with the highest degree of immunoreactivity should be chosen for analysis, and at least 1000 cells need to be analyzed in each case. The score of cells exhibiting staining in each case is evaluated semiquantitatively. A numeric intensity score is set from 1 to 4 (1 for no, 2 for weak, 3 for moderate, and 4 for strong staining). The fraction score (0–100 %) is defi ned by the percentage of positive tumor cells per slide. Total score range of 0–400 is obtained by multiplying the intensity score and the fraction score. The scores can be used to conduct statistical analysis as both continuous and binary variable by defining high and low expression levels.

2.

Here, we described the use of FITC-conjugated secondary antibody and propidium iodide for staining. Other fluorescein (e.g., rhodamine)-labeled antibodies and nuclei staining dye (e.g., DAPI) can also be used.

3.

Ectopic and knockdown expression should be performed in eIF3 low and high expression cells, respectively. For example, immortalized non-cancer cell lines such as NIH3T3, RIE, IEC, and IMR-90 cells with low eIF3a expression can be used to establish eIF3a overexpression cell lines, while cancer cell lines such as H1299, A549, HeLa, and MCF7 cells with high endogenous eIF3a expression can be used to knockdown eIF3a expression [11, 13, 14, 27, 28, 37].

4.

It is noteworthy that in vivo studies using animal models need to be approved by institutional animal care and use committee before initiation of animal study.

5.

Since polysome profiling enables analysis of the translational level of a large number of mRNAs, high-throughput methods should be used to detect these mRNAs. Previously, microarray is the major method to conduct the high-throughput analysis [6, 38]. With the rapid development of next-generation sequencing, RNA-seq is replacing microarray and becoming a powerful method to analyze polysome profiling. It is, thus, recommended.

6.

For supershift and competition, 2 μL of specific antibodies against target eIF3 proteins or 100-fold cold probe is added to the reaction mixture and incubate for 30 min before adding α[32P]-labeled probe.

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