Extended Data Fig. 3: zDHHC8 is required for GPX4 palmitoylation.
a,b, Immunoblotting analysis of GPX4 palmitoylation by ABE assay in HEK293T cells transfected with plasmids encoding indicated zDHHCs-Flag for 36 h. c, Immunoblotting analysis of zDHHC8 in mock or ZDHHC8-KO HT-1080 cells. d, Immunoblotting analysis of zDHHC8 in Zdhhc8-knockdown B16-F10 cells. e, The cell viability of mock or ZDHHC8-KO HT-1080 cells treated with RSL3 (0.05 μM) together with or without DFO (5 μM), Liproxstatin-1 (5 μM), Z-VAD-FMK (10 μM) or NSA (2.5 μM) for 24 h. f, LDH release measurement of mock or ZDHHC8-KO HT-1080 cells treated with RSL3 (0.05 μM) together with or without ferrostatin-1 (Fer-1, 2 μM), DFO (5 μM), Liproxstatin-1 (5 μM), Z-VAD-FMK (10 μM) or NSA (2.5 μM) for 16 h. g, Immunoblotting analysis of zDHHC8 in ZDHHC8-knockdown HT-1080 cells, MC38 cells and CRC derived organoid. h, The cell viability of shCtrl or shZDHHC8 HT-1080 cells treated with RSL3 (0.05 μM), ML162 (0.05 μM) or IKE (1 μM), together with or without Fer-1 (2 μM) for 24 h. i,j, The cell viability of Zdhhc8-knockdown (shZdhhc8) B16-F10 cells (i) and MC38 cells (j) treated with RSL3 (0.05 μM), ML162 (0.05 μM) or IKE (1 μM), together with or without Fer-1 (2 μM) for 24 h. k, The cell viability of mock or ZDHHC8-KO HT-1080 cells treated with the indicated concentrations of RSL3 (left) or ML162 (right) in conjunction with DMSO or 2-BP (10 μM) for 24 h. l, Relative GSH level of HT-1080 cells after treated with vehicle, IKE (1 μM) or indicated concentration of 2-BP for 12 h. Data in a-d,g are representative of three independent experiments. For e,h-k data are presented as mean ± s.d. of n = 4 biological replicates per group. For f,l, data are presented as mean ± s.d. of n = 3 biological replicates per group. Statistical analysis was performed using one-way ANOVA with Dunnett’s test (l) and two-way ANOVA with Tukey’s test (e,f,h-k); ns, not significant.
