Fig. 4.

Cystine promotes mTORC1 activation via GCN2-ATF4-SESN2 axis. a Heatmap of genes that were significantly upregulated or downregulated by cystine in either HCT116 or RKO. SESN2 was downregulated by cystine in both cell lines. b SESN2 mRNA levels were dramatically downregulated by cystine in both HCT116 and RKO cell lines, as measured by quantitative real-time PCR. Cells were cultured for 24 or 48 h in conditional media with 0, 25, or 200 μM cystine. β-Actin was used as the loading control. P-value was determined by one-way analysis of variance. c SESN2 protein levels were dramatically downregulated by cystine in both HCT116 and RKO cell lines, as measured by WB. d SESN2 protein levels were downregulated by cystine in RKO xenografts, as shown in Fig. 3g. Six samples were analyzed by WB. e Schematic mechanism shows the mechanism by which cystine negatively regulates SESN2 transcription and promotes mTORC1 activation. f Cystine reduced ATF4 level in nuclear fraction. Cells were cultured for 24 h in conditional media with 0, 25, or 200 μM cystine. Cell nuclear and cytoplasmic lysate were separated and subjected to WB analysis. g Cystine promotes mTORC1 activation via GCN2-ATF4-SESN2 axis. WB analysis was performed to detect p-GCN2/GCN2, ATF4, SESN2, and p-p70S6K/p70S6K protein expression in HCT116 and RKO cells cultured with conditioned media. h ATF4 depletion blocked cystine deprivation-induced mTORC1 inactivation. HCT116 and RKO cells were cultured for 24 h in conditional media with 200, 25, or 0 μM cystine and cell lysates were collected for WB analysis to detect protein expression. Data are shown as mean ± SD (b)