Fig. 8. ZDHHC20L is an effector of the LORD pathway.

a–d Incorporation of ³H-palmitic acid (³H-palm) in GPX4 from U2OS cells silenced for 72 h with pooled (a) or individual (c) ZDHHC siRNAs. Cells were metabolically labeled for 3 h at 37 °C with ³H-palm; GPX4 was immunoprecipitated (IP-GPX4), resolved by SDS–PAGE, and analyzed by Western blot (WB) and autoradiography (³H-palm). The levels of ³H-palm incorporation in siControl (Ctrl) were set to 100% and results are mean ± SEM, and each dot represents an independent experiment (n = 3). p values compare siCtrl, two-way ANOVA, Dunnett’s test. e HEK WT or ZDHHC20-KO cells were incubated with 100 μM C16:0-azide for the indicated times or with palmitic acid for 8 h. Incorporated C16:0-azide was detected by click chemistry with alkyne-mPEG 5 K (200 μM), followed by SDS–PAGE and WB for GPX4. Results are representative of three independent experiments. f–h same as in (a), for (f), HEK WT (Ctrl) or ZDHHC20-KO cells, recomplemented with empty vector or short/long ZDHHC20 or (h) U2OS cells left untreated or treated for 4 h with 50 μM H₂O₂. For f Myc-ZDHHC20 expression was confirmed by WB. The levels of ³H-palm incorporation were set to 100% in Control-WT cells for g, or untreated control cells, in (h). Results are mean ± SEM, and each dot represents an independent experiment (g) n = 4 and h n = 6). p values comparing to Ctrl or as indicated by, (g) two-way ANOVA, Tukey’s test, and (h) Two-tailed Student’s t-test. i Cellular glutathione measured in U2OS cells, siCtrl or siZDHHC20 (72 h) alone or recomplemented with short/long ZDHHC20 (24 h). Data are mean ± SEM from one of three independent experiments (n = 6). p values by, two-way ANOVA, Tukey’s test j Viability assessed using Promega Glo ATB detection in U2OS cells as in i, treated as in (h). Data are mean ± SEM from one of three independent experiments (n = 36 replicates). p values by, two-way ANOVA, Tukey’s test k Proposed Model: Under homeostasis, ATF2–SETDB1–KAP1–ZNF354A repress antioxidant genes. Lipid peroxide accumulation activates p38/JNK signaling, leading to ZNF354A dissociation and induction of antioxidant responses. Created in BioRender. abrami, I (https://BioRender.com/gvmrwgl).