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WIPI4-mediated ferroptosis is autophagy independent but ATG2 dependent. a, LDH release from SH-SY5Y cells following WIPI4-knockdown and WIPI2-knockdown via siRNA treatment (siWIPI4 and siWIPI2, respectively) in comparison to control cells (n = 3). b, LDH release from siWIPI4- and scramble siRNA (scr)-treated ATG16L1-KO and control (wild-type, WT) HeLa cells (n = 3). c, Rod photoreceptor area of rho:EGFP zebrafish retinas at 10 d.p.f. showing the autophagy-independent effect of wdr45-targeting CRISPR knockdown via injection with wdr45-targeting CRISPR guides (wdr45 KD). We analysed cryosections across fish retinas of uninjected zebrafish larvae and their siblings injected with either wdr45 KD or atg7-targeting CRISPR guides (atg7 KD), or both in combination, followed by treatment with DMSO or 10 μM Fer-1 at 5–10 d.p.f. Data are the mean ± s.d.; n = 22 eyes per group; two-tailed unpaired Student’s t-test. d, Representative images of rho:EGFP zebrafish retinas at 10 d.p.f. showing the autophagy-independent effect of wdr45 KD. Scale bar, 50 μm. e, ATG2A/B WT or CRISPR KO (ATG2A/B KO) HeLa cells were transfected with control siRNA or siWIPI4 for 72 h and subjected to an LDH assay (n = 3). f, MDA analysis of the cells in e (n = 3). Controls normalised to 100. g, Cell viability of ATG2A/B KO HeLa cells transfected with GFP or GFP–ATG2A for 24 h and treated with 100 nM Lip-1 or 1 µM Fer-1 for 20 h following a medium change (n = 3). h, LDH release from control and WIPI4-knockdown HeLa cells transfected with GFP, GFP–WIPI4 WT or LOOP3 siRNA-resistant mutant (ΔLOOP3; n = 4). a,b,e–h, Data are the normalized mean ± s.d.; n, number of biologically independent experiments; two-tailed one-sample Student’s t-test for comparisons to the control and two-tailed paired Student’s t-test for comparisons between other samples. *P < 0.05 and **P < 0.01. Source numerical data are provided. Source data
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