Fig. 3.

Vitamin B₁₂ depletion induces changes in γH2AX, a marker of DNA damage in HeLa cells. Cells were stained for DNA content (Vybrant Violet; 1–4, A) and γH2AX (FITC; 1–4, B–E); individual plots depict the cell count (y axis) vs. fluorescence intensity (x axis). The high γH2AX parameter is a threshold defined by the mean top 2.5% of cells in G1, S, and G2/M (all cells) stained for γH2AX in the vitamin B₁₂- and folate-replete condition (1, B), and this gate was uniformly applied to all conditions and cell cycle phases. Each plot shows the mean percent high γH2AX ± SD for triplicate measurements for each experimental condition and cell cycle phase. Individual triplicate measurements for each condition are plotted relative to the corresponding mean γH2AX values in the vitamin B₁₂- and folate-replete condition (hatched histograms, 1, B–E). Asterisks designate statistical significance in percent high γH2AX values between treatment conditions and cell cycle phases compared with the corresponding phases in the vitamin B₁₂- and folate-replete condition (SI Appendix, Table S1). The greatest percentage of cells with high γH2AX within conditions was observed in G2/M under vitamin B₁₂- and folate-depleted conditions (P < 0.001; 4, E). The vitamin B₁₂-depleted and folate-replete condition in row 3 contains duplicate measurements. The experiment was performed twice (SI Appendix, Fig. S3). Folate-replete, 25 nM (6S) 5-formylTHF in culture media; folate-depleted, 5 nM (6S) 5-formylTHF in culture media. The statistical significance is represented as follows: NS, not significant (P > 0.05); *P = 0.01 < P < 0.05; **P = 0.01 < P < 0.001; ***P < 0.001.