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. 2020 Aug 6;11(8):649. doi: 10.1038/s41419-020-02825-y

Fig. 5. Acetylation of MTHFD2 at K88 restrains cellular redox balance.

Fig. 5

a Identification of HCT116 MTHFD2 rescued cell lines. MTHFD2 was knocked out in HCT116 cells, empty vector, Flag-tagged WT, and 88 K to Q mutant of MTHFD2 was stably re-expressed in MTHFD2 KO cells. Total lysates were prepared from four cell lines and detected by western blot analysis. b MTHFD2 K88Q decreases cellular NADPH level under cisplatin treatment. HCT116 WT cells, HCT116 KO cells, MTHFD2 WT cells, or MTHFD2 K88Q cells were treated in the presence or absence of cisplatin for 24 h. Cell’s cellular NADPH level was measured. Error bars, ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., not significant for the indicated comparison. c MTHFD2 K88Q decreases cellular GSH level under cisplatin treatment. HCT116 WT cells, HCT116 KO cells, MTHFD2 WT cells, or MTHFD2 K88Q cells were treated in the presence or absence of cisplatin for 24 h. GSH level in cells was measured. Error bars, ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., not significant for the indicated comparison. d MTHFD2 K88Q increases cellular ROS level under cisplatin treatment. HCT116 WT cells, HCT116 KO cells, MTHFD2 WT cells, or MTHFD2 K88Q cells were treated in the presence or absence of cisplatin for 24 h and ROS level was measured. Error bars, ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., not significant for the indicated comparison. e Identification of SIRT3 overexpression cell lines. Empty vector, HA-tagged SIRT3 was stably re-expressed in HCT116 cells and MTHFD2 KO cells. Total lysates were prepared from four cell lines and detected by western blot analysis. f SIRT3 rescues cellular NADPH level under cisplatin treatment. HCT116 cells, SIRT3 overexpression cells, SIRT3 overexpression-MTHFD2 KO cells, or MTHFD2 KO cells were treated in the presence or absence of cisplatin for 24 h. Cell’s cellular NADPH level was measured. Error bars, ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., not significant for the indicated comparison. g SIRT3 rescues cellular GSH level under cisplatin treatment. HCT116 cells, SIRT3 overexpression cells, SIRT3 overexpression-MTHFD2 KO cells, or MTHFD2 KO cells were treated in the presence or absence of cisplatin for 24 h. GSH level in cells was measured. Error bars, ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., not significant for the indicated comparison. h SIRT3 rescues the higher cellular ROS under cisplatin treatment. HCT116 cells, SIRT3 overexpression cells, SIRT3 overexpression-MTHFD2 KO cells, or MTHFD2 KO cells were treated in the presence or absence of cisplatin for 24 h and ROS level was measured. Error bars, ± SD (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n.s., not significant for the indicated comparison.