Fig. 7: Depletion of dTMP induces cell death while inhibition of purine synthesis induces growth and cell cycle arrest.
From: Formate overflow drives toxic folate trapping in MTHFD1 inhibited cancer cells
a–c, Proliferation of (a,b) SW620 WT and (c) MTHFD2−/− cells measured as fold change of cell confluence after (a) 1 μM TH9619 or TH9975 (b,c) with or without 50 μM hypoxanthine and 50 μM thymidine (cultured in RPMI-FBS or RPMI-dFBS). AUC of proliferation curves is shown as means ± standard error ((a) n = 3 for WT, n = 4 for MTHFD2−/− (b,c) n = 4); one-way ANOVA with (a) Šídák’s multiple comparisons test or (b,c) Tukey’s multiple comparisons test. d,e, Cell cycle analysis of SW620 WT and MTHFD2−/− cells treated with TH9619, 50 μM thymidine (T), 250 μM hypoxanthine (H) or vehicle (cultured in (d) RPMI-FBS or (e) RPMI-dFBS), means ± s.d. (n = 3); one-way ANOVA with Tukey’s multiple comparisons test for S-phase. f–h, Cell death analysis by annexinV/PI staining of SW620 WT and MTHFD2−/− cells after 96 h treatment with 1 μM TH9619 or TH9975 and (f,g) 50 μM hypoxanthine (H) and 50 μM thymidine (T) cultured in (f) RPMI-FBS or (g) RPMI-dFBS, or (h) treated with T and 1 mM formate (F) cultured in RPMI-dFBS, means ± s.d. (n = 3); one-way ANOVA analysis with Tukey’s multiple comparisons test for pooled early and late apoptotic populations. i, MTHFD2 protein levels in SW620, MDA-MB468 and MDA-MB-231 cells normalized to β-actin, means ± s.d. (n = 3); one-way ANOVA analysis with Tukey’s multiple comparisons test. j, Formate release rates of SW620, MDA-MB-468 and MDA-MB-231 cells, means ± s.d. (n = 7 for SW620, n = 12 for MDA-MB-468 and n = 3 for MDA-MB-231). k, Proliferation of MDA-MB-468 WT and MTHFD2−/− cells and MDA-MB-231 cells measured as fold change of cell confluence in response to 1 μM TH9619 and 50 μM hypoxanthine and 1 mM sodium formate (cultured in RPMI-dFBS). AUC of curves is shown as means ± s.e. (n = 3 for MDA-MB-468 and n = 4 for MDA-MB-231); one-way ANOVA with Tukey’s multiple comparisons test. l, Cell death analysis of MDA-MB-468 WT and MTHFD2−/− cells after 96 h treatment with 1 μM TH9619 and 50 μM hypoxanthine (H) and 1 mM formate (F) (cultured in RPMI-dFBS), means ± s.d. (n = 4); one-way ANOVA analysis with Tukey’s multiple comparisons test for pooled early and late apoptotic populations.
