Fig. 3: GPX4 palmitoylation regulates protein stability.

Immunoblots of GPX4, FSP1, SLC7A11, and ACSL4 in A375 and A549 cells treated with varying concentrations of 2-BP (a) and at different time points (b). c Immunofluorescence staining of GPX4 in A375 cells treated with 2-BP for 48 h. Scale bar, 10 μm. d, e Immunoblots of GPX4 in A375 cells pre-treated with 2-BP for 24 h, followed by cycloheximide (CHX) treatment for indicated times (d). Quantification of GPX4 protein levels (e). f GPX4 detected by immunoblots in A375 cells pre-treated with 2-BP for 24 h, followed by 12 h proteasome inhibitor MG132 treatment. g GPX4 detected by immunoblots in A375 cells pre-treated with 2-BP for 24 h, followed by 12 h lysosome inhibitor BafA1 treatment. h Immunoblots were performed on A375 cells that were pre-treated with 2-BP for 12 h, followed by the addition of 10 μM MG132 for an additional 12 h, after which GPX4 was pulled down using an anti-GPX4 antibody. i, j Immunoblots of GPX4 in A375 cells infected with Flag-GPX4-WT or Flag-GPX4-C66S, followed by CHX treatment for indicated times (i). Quantification of GPX4 protein levels (j). k, l Immunoblots of GPX4 in A375 cells infected with Flag-GPX4-WT or Flag-GPX4-C75S, followed by CHX treatment for indicated times (k). Quantification of GPX4 protein levels (l). GPX4 protein levels detected by immunoblots (m) and relative mRNA levels by RT-qPCR (n) in A375 cells infected with various GPX4 constructs. o GPX4 detected by immunoblots in A375 cells infected with Flag-GPX4-WT or Flag-GPX4-C66S, treated with or without MG132 for 12 h. p, q Immunoblots of GPX4 in A375 cells infected with various GPX4 constructs, followed by 48 h 2-BP treatment (p). Quantification of GPX4 protein levels (q). Data are presented as mean ± SD. Statistical analysis: two-way ANOVA with Tukey’s test for (q); one-way ANOVA with Tukey’s test for (e, j, l, n). Sample sizes: (e, j, l, n, q) n = 3 independent experiments. Image in a–d, f–i, k, m, o, p shows representative results from three independent experiments.