Fig. 4: ZDHHC20 is the palmitoyl acyltransferase of GPX4.

a Palmitoylation of GPX4 in 293T cells expressing Flag-GPX4 and transfected with either empty vector or HA–DHHC plasmids, incubated with 50 μM Alk14 for 12 h, determined using the click chemistry assay. b Co-immunoprecipitation in 293T cells transiently transfected with Flag-GPX4 and HA-ZDHHC20 plasmids to investigate ZDHHC20-GPX4 interaction, with GPX4 and ZDHHC20 detection by immunoblots. RT-qPCR analysis of ZDHHC20 (c) and GPX4 (d) mRNA levels in A375 cells infected with lentiviruses encoding control shRNA or ZDHHC20 shRNA. e GPX4 palmitoylation in A375 cells infected with lentiviruses encoding control shRNA or ZDHHC20 shRNA, and incubated with 50 μM Alk14 for 12 h, determined using the click chemistry assay. f GPX4 palmitoylation in A375 cells infected with lentiviruses encoding HA-empty vector, HA-ZDHHC20 or HA-ZDHHC20-C156S and incubated with 50 μM Alk14 for 12 h, determined using the click chemistry assay. g Cell viability analysis in A375 cells infected with lentiviruses encoding control shRNA or ZDHHC20 shRNA, followed by 48 h RSL3 treatment. h Immunoblots of GPX4 and ZDHHC20 in A375 cells infected with lentiviruses encoding empty vector or ZDHHC20. i Cell viability analysis in A375 cells infected with lentiviruses encoding empty vector or ZDHHC20, followed by 24 h RSL3 treatment. j Immunofluorescence staining for GPX4 and ZDHHC20 was performed in A375 cells infected with lentiviruses encoding a control plasmid or an HA-ZDHHC20 plasmid. Scale bar, 4 μm. k Analysis of the correlation between ZDHHC20 gene expression and ferroptosis sensitivity to RSL3, ML162 and ML210 using the CTRP database (https://portals.broadinstitute.org/ctrp.v2.1/). ZDHHC20 was correlated with resistance to RSL3 (P = 0.139), ML162 (P = 0.135) and ML210 (P = 0.154) according to their Pearson correlation score. “In Fig. 4k, the box plot is presented to illustrate the correlation between the sensitivity of [860 cancer cell lines to 481 compounds] and [different levels of ZDHHC20 gene expression]. The central box extends from the lower quartile (-1, Q1, 25th percentile) to the upper quartile (1, Q3, 75th percentile), indicating the interquartile range (2, IQR), which contains the middle 50% of the data. The median (0, Q2, 50th percentile) is represented by a line inside the box, dividing the data into two halves. The whiskers extend from the edges of the box to the minimum and maximum data points within a certain range, typically 1.5 times the IQR, unless there are outliers. Outliers are data points that fall outside the range of -3 to 3 and are plotted as individual points marked by circles. Data are presented as mean ± SD. Statistical analysis: one-way ANOVA with Tukey’s test for (c, d). Sample sizes: (c, d, g, i) n = 3 independent experiments. k n = 860 cancer cell lines. Image in a, b, e, f, h, j shows representative results from three independent experiments.