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. 2017 Mar 6;114(12):E2319–E2326. doi: 10.1073/pnas.1619745114

Fig. 4.

Fig. 4.

Low-dose As2O3 exposure inhibits de novo thymidylate biosynthesis. (A) As2O3 induced depletion of SHMT1 and MTHFD1 proteins in a time-dependent manner. HeLa cells were treated with 5 μM As2O3 for indicated periods of time before cell lysis. Cell lysates (WCL) were probed with α-MTHFD1 and α-SHMT1 antibodies by immunoblotting. Actin was used to confirm equal sample loading (SI Appendix, Fig. S3A). (B) Low-dose As2O3 results in suppression of de novo dTMP synthesis in a dose-dependent manner. The dU suppression assay was used to measure the contribution of the de novo dTMP synthesis pathway (as indicated by [14C]-dU incorporation) relative to the dTMP salvage pathway (as indicated by [3H]-dT). (C and D) The effect of arsenic and folate on uracil levels in genomic DNA. Cells were cultured in modified MEM (modMEM) with [+Fol, 25 nM (6S) 5-formylTHF] or without [−Fol] (6S) 5-formylTHF in the culture medium and with and without 1 μM As2O3 treatment for 24 h in Hek293 (C) and HeLa (D) cells. (E and F) The effect of arsenic and folate on uracil levels in genomic DNA in αMEM containing 2.2 µM folic acid. Cells were cultured in αMEM with and without 1 μM As2O3 treatment for 24 h in HeLa (E) and Shmt1−/− MEFs (F). One micromole As2O3 treatment for 24 h resulted in an increase in genomic uracil in HeLa cells (E) and in Shmt1−/− MEFs (F) cultured in αMEM media containing folic acid and nucleosides. (G) The effect of arsenic and folate on nuclear area with γH2AX, a marker of DNA damage. Cells were cultured in modified MEM (modMEM) with [+Fol, 25 nM (6S) 5-formylTHF] or without (−Fol) (6S) 5-formylTHF in the culture medium and with and without 1 μM As2O3 treatment for 24 h in HeLa cells. Folate-deficient media and 1 μM As2O3 treatment for 24 h increased γH2AX in HeLa cells. (H and I) As2O3 induces nuclear γH2AX foci and intensity in a dose-dependent manner. Data are presented as mean ± SEM. Statistical significance was assessed by Student t test in B, C, D, E, and F, and Mann–Whitney/Wilcoxon test in G, H, and I, with Bonferroni correction. NS P ≥ 0.05, *0.01 < P < 0.05, **0.001 < P < 0.01, ***P < 0.001.