Figure 3(A-B) IDH1 K224 acetylation level was enhanced with increasing glucose concentration. Flag-IDH1 was overexpressed in cells treated with increased concentrations of glucose for 6 h. IDH1 proteins were purified by Flag beads, and then IDH1 K224 acetylation level was determined by Western blot and IDH1 catalytic activity was assessed. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For B statistical significance was assessed with the one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01. n.s. = not significant.(C-D) IDH1 K224 acetylation level was upregulated and IDH1 activity was weakened with increasing glutamine concentration. Flag-IDH1 was overexpressed in cells treated with increased glutamine for 6 h. IDH1 proteins were purified by Flag beads, and the K224 acetylation level of IDH1 was determined by Western blot analysis and then IDH1 catalytic activity was evaluated. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For , D statistical significance was assessed with the one-way ANOVA with Newman-Keuls post hoc test. P<0.05 and **P<0.01. n.s. = not significant.Downregulation of SIRT2 diminished the effect of glucose on changing IDH1 K224 acetylation. Flag-tagged IDH1 was overexpressed in HCT116 cells with or without transient SIRT2 knockdown. The cells were treated with different concentrations of glucose (E)Downregulation of SIRT2 diminished the effect of glutamine on changing IDH1 K224 acetylation. Flag-tagged IDH1 was overexpressed in HCT116 cells with or without transient SIRT2 knockdown. The cells were treated with different concentrations of glutamine (F).(G) IDH1 K224 deacetylation regulated cellular NADPH/NADP+ redox in cells. In HCT116 stable cells with IDH1 knockout and re-expressing the indicated proteins, the ratio of NADPH/NADP+ in cells was measured as followed by manufacture's instruction. Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For , G statistical significance was assessed with the one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01. n.s. = not significant.(H) IDH1 K224 deacetylation promoted GSH production in HCT116 cells. In HCT116 stable cells with IDH1 knockout and re-expressing the indicated proteins, the ratio of GSH/GSSG was assessed as followed by manufacture's instruction. 1 Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For H, statistical significance was assessed with the one-way ANOVA with Newman-Keuls post hoc test. *P<0.05 and **P<0.01. n.s. = not significant.(I) IDH1 K224 deacetylation suppressed cellular ROS levels in HCT116 cells. ROS was determined in cells under non-stressed condition or exposed to menadione. *P<0.05 and **P<0.01 Data information: All results were expressed as mean ± SD of three independent experiments (n ≥ 3 per experimental condition). For figure I, statistical significance was assessed with the two-way ANOVA followed by Tukey's post hoc test. *P<0.05 and **P<0.01. n.s. = not significant.