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. 2023 Aug 8;14:4758. doi: 10.1038/s41467-023-40518-0

Fig. 3. CHAC1 induced by cystine deprivation accelerates GSH depletion and facilitates ferroptosis.

Fig. 3

a Venn diagram showing common genes that are upregulated by cystine deprivation and erastin treatment and belong to the geneset of GOBP—glutathione metabolic process. b Relative mRNA level of CHAC1 in HT-1080 cells treated with cystine deprivation (left) or 1 μM IKE (right) at different time points. c Immunoblot of human CHAC1 in HT-1080 cells treated with cystine deprivation (upper) or IKE (lower) at indicated time points. Tubulin serves as a loading control. d, e HT-1080 cells expressing control guide RNA (sgControl) or CHAC1 guide RNA (sgCHAC1) were treated with cystine deprivation or 5 μM erastin. Total GSH content (d) was measured at 8 h, and cell death (e) was measured at 16 h (for Cyss−) or 20 h (for IKE). f Immunoblot of human CHAC1 in HT-1080 cells expressing scramble shRNA or three different CHAC1 shRNA (shCHAC1–1/2/3), and GAPDH serves as loading control. g Cell death of HT-1080 cells expressing scramble or CHAC1 shRNA in response to cystine deprivation (left) or 5 μM erastin treatment (right) for 17 h. h Cell death of Hepa1–6 cells expressing sgControl or two different sgCHAC1 under 5 μM erastin treatment for 20 h was quantified (h). i Immunoblots of phosphorylated eIF2α (p-eIF2α), total eIF2α and ATF4 in HT-1080 cells treated by cystine deprivation for indicated time points. Tubulin serves as a loading control. j Immunoblot of CHAC1 in scramble or shATF4 expressing HT-1080 cells in response to cystine deprivation for 8 h. GAPDH serves as loading control. k Cell death of HT-1080 cells expressing scramble or shATF4 in response to cystine deprivation, IKE (1 μM), or erastin (5 μM) for 18 h. l Cell death of HT-1080 cells treated by cystine deprivation plus GCN2 inhibitor (GCN2iB, 1 μM) for 15 h. The experiment was repeated twice with similar results (c, i). n = 3 biological replicates and P values are determined by two-way ANOVA (d, e, g–k) or one-way ANOVA (l). Source data are provided as a Source Data file.