Figure 5. Metabolic characterization of MLYCD depletion.

(A) Schematic representation of isotope tracing experiment with ¹³C₁₆-Palmitate. Black-filled circles indicate ¹³C-carbon, whereas the white filled circles represent the unlabeled carbon. The schematic shows the expected composition of labeled carbons of the indicated metabolites. (B) Labeling incorporation from ¹³C-Palmitate into Palmitoyl-carnitine in non-targeting control (NTC) and MLYCD-depleted (shMLYCD) cells. Data are shown as percentage of ¹³C₁₆-palmitoylcarnitine to the total pool of Palmitoyl-carnitine. (C) Labeling incorporation from ¹³C₁₆-palmitate into TCA cycle intermediates of the indicated cell lines. Data are shown as percentage of the m+2 isotopologue to the total pool size of each metabolite. (D) Schematic representation of isotope tracing experiment with ¹³C₆-Glucose. The distribution of light and heavy carbons is depicted as in A. (E) Labeling of Citrate and of (F) Palmitate after incubation with ¹³C₆-glucose. Data are shown as percentage of the indicated isotopologue to the total pool size of each metabolite. Isotopologue distribution of citrate is indicated in Figure 5—figure supplement 6. Palmitate isotopologues above m+10 were not detected (G) Schematic representation of isotope tracing experiment with 1,2-¹³C₂-Glucose. Ru5p: ribulose-5-phosphate. The distribution of light and heavy carbons is depicted as in A. (H) Ratio between m+1 and m+2 isotopologues of Lactate in the indicated cell lines. (I) Ratio between reduced (GSH) and oxidized (GSSG) glutathione in RPMI-8226 cells infected with the indicated constructs. (J) Cell counts after 72 hr of culture of the indicated cell lines in the presence or absence of 2 mM N-Acetyl Cysteine. Data are shown as mean ± s.e.m of three independent cultures. *p-value<0.05. **p-value<0.01. ***p-value < 0.001.

DOI: http://dx.doi.org/10.7554/eLife.03641.010

Figure 5—figure supplement 1. Silencing of MLYCD in RPMI-8226 cells using shRNA.

MLYCD mRNA expression upon infection with Non Targeting Control (NTC) and two independent shRNA constructs targeting MLYCD (shMLYCD1 and 2) in RPMI-8226 cells.

DOI: http://dx.doi.org/10.7554/eLife.03641.011

Figure 5—figure supplement 2. Effects of Silencing of MLYCD in RPMI-8226 cells.

Cells were treated as indicated in Figure 5—figure supplement 1 and counted after 72 hr of culture were.

DOI: http://dx.doi.org/10.7554/eLife.03641.012

Figure 5—figure supplement 3. Isotopologue distribution of TCA cycle intermediates after incubation with ¹³C₁₆-palmitate.

Labeling incorporation from ¹³C₁₆-palmitate in NTC and shMLYCD2 cells. Isotopologues above m+2 were not detected. Data are presented as percentage of the indicated isotopologue to the total pool of each metabolite. Data are presented as mean ± s.e.m.

DOI: http://dx.doi.org/10.7554/eLife.03641.013

Figure 5—figure supplement 4. LCMS analyses of TCA cycle intermediates in MLYCD-depleted cells.

Total levels of TCA cycle intermediates in RPMI-8226 cells infected with NTC or shMLYCD2. Data are presented as relative abundance of total metabolites in shMLYCD2 compared to NTC.

DOI: http://dx.doi.org/10.7554/eLife.03641.014

Figure 5—figure supplement 5. A schematic description of the metabolic changes following MLYCD knock down.

MLYCD suppression is predicted to divert the accumulated malonyl-CoA to fatty acid biosynthesis, increasing the demand of cells for reducing power. The latter is generated by the oxidative branch of the pentose phosphate pathway, overall leading to increased oxidative stress. Green/red arrows represent increased/decreased flux, respectively.

DOI: http://dx.doi.org/10.7554/eLife.03641.015

Figure 5—figure supplement 6. TCA cycle activity in MLYCD-depleted cells.

Labeling incorporation from ¹³C₆-Glucose in NTC and shMLYCD2 cells. Data are presented as percentage of the indicated isotopologue to the total pool of each metabolite. Data are presented as mean ± s.e.m.

DOI: http://dx.doi.org/10.7554/eLife.03641.016