Fig. 5.

SHMT1 and MTHFD1 protein expression modulate genome stability. (A) Reducing SHMT1, SHMT2, and MTHFD1 expression increases genome instability, shown as percentage nuclear area with γH2AX foci in HeLa cells treated with validated siRNAs. The P values (Mann–Whitney/Wilcoxon test) are presented for comparisons with the control condition (siCON) with scrambled siRNAs. (B) Reducing SHMT1, SHMT2, and MTHFD1 expression increases genome instability, shown as relative integrated γH2AX fluorescence intensity presented as mean per nucleus ± SEM in HeLa cells treated with siRNA as in A. The P values (Mann–Whitney/Wilcoxon test) are presented for comparisons with the control condition (siCON) with scrambled siRNAs. Experiments were repeated three times. (C) Representative SHMT1, SHMT2, and MTHFD1 immunoblots confirm the efficacy of siRNA treatments in A and B. Actin was used to confirm equal loading (SI Appendix, Fig. S3). (D) The effect of SHMT1 expression in MEFs on genome stability. MEFs were cultured for 24 h in αMEM medium containing 4 µM As₂O₃ before fixation_. The percentage nuclear area with γH2AX foci was measured as in A. (E) Representative confocal microscopy images used for γH2AX quantifications in siRNA-treated HeLa cells in A and B. draq5 (blue, Middle) was used as nuclear stain. Ten and 20 images per condition were acquired and analyzed using Metamorph software. (F) Relative integrated γH2AX fluorescence intensity presented as mean per nucleus ± SEM in MEF cells treated as in D. P values (Mann–Whitney/Wilcoxon test with Bonferroni correction) are graphed as follows: nonsignificant, NS P ≥ 0.05, *0.01 < P < 0.05, **0.001 < P < 0.01, ***P < 0.001.