Extended Data Fig. 9: Investigating metabolic consequence of Complex III impairment and activity of paralogous pathways other than oxidative MTHFD2.
(a) Effect of MTHFD2 inhibitor (10 µM) and uridine (50 µM) supplementation on viability of parental ovarian cancer lines that are UQCR11-intact (SKOV3) or UQCR11-null (HeyA8, OVCAR8, CAOV3). Data are representative of two independent experiments and presented as mean ± s.d. (n=6). Data analyzed with one-way ANOVA with Tukey’s correction for multiple comparisons. (b) Effect of MTHFD2 inhibitor and uridine supplementation on viability of isogenic SKOV3 lines expressing shScramble or shUQCR11. Data are representative of two independent experiments and presented as mean ± s.d. (n=6). Data analyzed with one-way ANOVA with Tukey’s correction for multiple comparisons. (c) Viability of SKOV3 and OVCAR8 cells expressing dox-inducible shScramble or shMTHFD2 supplemented with pyruvate. Data are representative of two independent experiments and presented as mean ± s.e.m. (n=6) and analyzed using two-tailed Student’s t-test. (d) mRNA expression of NNT, IDH2, IDH3A measured via qRT-PCR after knocking down with shNNT, shIDH2, and shIDH3A, relative to scramble shRNA (shScr), verifying knockdown efficiency in SKOV3 and OVCAR8 cell-lines. Data are representative of two independent experiments and presented as mean ± s.d. (n=4). Data analyzed using unpaired two-tailed Student’s t-test. (e) Viability of SKOV3 and OVCAR8 cells after transfection of shNNT, shIDH2, and shIDH3A, relative to respective SKOV3 and OVCAR8 controls transfected with scramble shRNA (shScr). Data are representative of two independent experiments and presented as mean ± s.d. (n=5). Data are analyzed using ordinary one-way ANOVA with multiple comparisons. (f) SKOV3 and OVCAR8 cells transfected with shScramble, shNNT, shIDH2, and shIDH3A treated with MTHFD2 inhibitor (10 µM) for 72 h. Loss of viability calculated by comparing respective viability in vehicle control-treated cells to MTHFD2 inhibitor-treated cells. Data are representative of two independent experiments and presented as mean ± s.e.m. (n=6) and analyzed using ordinary one-way ANOVA with Dunnett’s correction for multiple comparisons. Error bars are calculated using propagation of error principles.
