WIPI4 loss of function induces ferroptosis.

a, LDH release, as a measure of cytotoxicity (n = 5), from control and WIPI4-knockdown SH-SY5Y cells following treatment with dimethylsulfoxide (DMSO; control), 1 µM Z-VAD-fmk (Z-VAD), 10 µM necrostatin-1 (Nec-1), 100 nM liproxstatin-1 (Lip-1) or 1 µM Fer-1 for 48 h. Unless specified otherwise, oligonucleotide J-019758 was used as the siWIPI4 in all experiments. b, Cell viability, measured using AquaBluer (n = 3), of control and WIPI4-knockdown SH-SY5Y cells treated as in a. c, HeLa cells transiently transfected with pSpCas9(BB)-2A-GFP with single guide RNA (sgRNA) targeting WIPI4 exon 3 (sgWIPI4) or its control construct were cultured for 24 h before treatment with N-acetyl-l-cysteine (NAC) or deferoxamine (DFO), at the indicated concentrations, in media containing IncuCyte red dye. The cells were imaged 72 h post transfection. The number of dead cells were determined as the number of red objects per well normalized to that of the scramble and spCas9 control construct-transfected cells treated with DMSO (n = 3). d, Levels of MDA of control and WIPI4-knockdown SH-SY5Y cells treated with DMSO or 1 µM Fer-1 for 24 h (n = 3). e, Images of BODIPY_581/591C11-stained i³ neurons. On Day 14 of differentiation, the i³ neurons were treated with scramble and anti-human WIPI4 smartpool shRNA (shWIPI4) delivered by PLKO.1 packaged in lentivirus for 24 h, followed by treatment with 5 µM Fer-1 or DMSO in fresh media without virus for another 24 h before imaging. Scale bars, 10 µm. f, Average ratio of the intensities of the 510 and 591 nm emissions quantified from the images in e (n = 5; ≥5 fields imaged per experiment). a–d,f, Data are the normalized mean ± s.d.; two-tailed one-sample Student’s t-test for comparisons with the control and two-tailed paired Student’s t-test for comparisons with other samples (a–d); two-tailed paired Student’s t-test (f); *P < 0.05 and **P < 0.01. Scr, scramble; n, number of biologically independent experiments. Source numerical data are provided.

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WIPI4 loss of function induces ferroptosis in zebrafish.

a, Zebrafish juveniles with wdr45 knockdown (wdr45 KD) by CRISPR injection had reduced lifespans compared with their uninjected siblings from the age of 4 weeks. Survival rate of WIPI4 CRISPR mutants and their uninjected siblings over a period of 7 weeks (n = 60 fish per group); log-rank Mantel–Cox test. b, Treatment of rho:EGFP transgenic fish with 10 μM Fer-1 from 5 to 10 d.p.f. rescued the loss of photoreceptors following wdr45 KD by CRISPR injection, whereas Fer-1 did not cause any change in the fluorescence rod area of uninjected fish. Representative images of sections across the eye of 10 d.p.f. rho:EGFP fish injected with wdr45-targeting CRISPRs and their uninjected siblings treated with DMSO or 10 μM Fer-1, respectively. Scale bar, 50 μm. c, Photoreceptor areas from the images in b; n ≥ 23 eyes per group. d, Increase in lipid peroxidation, represented by higher concentrations of MDA, in wild-type fish subjected to wdr45 KD by CRISPR injection at 5 d.p.f. compared with their uninjected siblings; n = 5 biologically independent experiments, 30 fish each. Data are the mean ± s.d. *P < 0.05 and **P < 0.01; two-tailed unpaired Student’s t-test. Source numerical data are provided.

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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.

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WIPI4 depletion mislocalizes ATG2.

a, Average duration of GFP–ATG2 and RFP–LC3 co-localization in HeLa cells stably expressing RFP–LC3 quantified from videos taken of living cells (n = 3 independent biological repeats; ≥3 co-localization events in different cells imaged per experiment). The maximum length of each video was 140 s. Data are the mean ± s.d.; two-tailed paired Student’s t-test. b, HeLa cells transfected with control siRNA or siWIPI4 were subjected to cell fractionation and immunoblotted with antibodies to the indicated proteins. For fractionation, isotonic buffer was used. Mitochondria were collected as pellets resuspended in PBS. This may explain the different mobility of WIPI4 in these different fractions. PNS, post-nuclear supernatant; mito, mitochondrial fraction; post-mito, post mitochondrial fraction. c, Pearson’s coefficient of endogenous ATG2A with calnexin in ATG16-KO and wild-type control cells (n = 3 biologically independent experiments; ≥20 fields imaged in each experiment). Data are the normalized mean ± s.d.; 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. d, ATG2A/B-WT and -KO HeLa cells transfected with control siRNA or siWIPI4 were immunoprecipitated (IP) with endogenous anti-TMEM41b and blotted for ATG2A and TOMM40. *P < 0.05 and **P < 0.01. Source numerical data and unprocessed blots are provided.

The ATG2–WIPI4 axis regulates ferroptosis dependent on mitochondria.

a, LDH release from HeLa cells transfected with the indicated siRNAs (n = 3). LDH release was measured 72 h post transfection (top). The western blots show the silencing efficiency of siWIPI4 and siRNA targeting TOMM40 (siTOMM40). b, Ratio of ATG2A levels in the mitochondrial versus post-mitochondrial fractions (n = 4). c, ATG2A/B-KO HeLa cells transfected with GFP, GFP–ATG2A-WT, GFP-ATG2A-ΔMLD or GFP-ATG2A-YFS were subjected to cell fractionation and immunoblotted. d, HeLa cells transfected with GFP, GFP–ATG2A-WT, GFP-ATG2A-ΔMLD or GFP-ATG2A-YFS were immunoprecipitated by GFP-Trap beads and blotted for TOMM40 and WIPI4. e, LDH release from ATG2A/B-KO HeLa cells transfected with GFP, GFP-ATG2A-WT or GFP-ATG2A-ΔMLD (n = 3). f, Levels of MDA in ATG2A/B-KO HeLa cells transfected with GFP, GFP-ATG2A-WT, GFP-ATG2A-ΔMLD or GFP-ATG2A-YFS (n = 4). Control normalised to 100. g, Lipid peroxidation evaluated by MitoPerOx staining of primary neurons (n = 3; ≥5 fields were imaged per sample). The peroxidation levels were calculated as the fluorescence intensities of peroxidized (510 nm emission) signal normalized to total signal (591 nm emission). The values were then normalized to that of scramble DMSO. Mouse primary neurons were cultured for five days before infection with scramble or anti-hWIPI4 smartpool shRNA delivered by lentivirus for 24 h, and then treated with 5 µM inPISD or DMSO in fresh media for one day. h, LDH release of SH-SY5Y cells transfected with control siRNA or siWIPI4 for 72 h, followed by incubation with the indicated concentration of MitoTEMPO (MT) for 48 h (n = 4). i, Mitophagy was induced in HeLa cells stably expressing PARKIN–FLAG with 12.5 µM CCCP for 48 h before transfection with siWIPI4. The cells were then cultured for 24 h in fresh media and LDH release was assessed (n = 3). a,b,e–i, Data are the normalized mean ± s.d.; 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; n, number of biologically independent experiments. WT, GFP–ATG2A-WT; ΔMLD, GFP-ATG2A-ΔMLD; YFS, GFP-ATG2A-YFS. *P < 0.05, **P < 0.01 and ***P < 0.001. Source numerical data and unprocessed blots are provided.

Mitochondrial PE levels are increased by ATG2 and depend on its lipid transfer function.

a–d, HeLa cells transfected with control siRNA or siWIPI4 were subjected to cell fractionation. Equal amounts of protein samples were then used for PE, PC and PS measurements (a, n = 4; b–d, n = 3). e, Unsaturated fatty acid (UFA) levels in the mitochondrial fractions and total lysates of HeLa cells transfected with siWIPI4 or scramble siRNA (n = 3). HeLa cells transiently transfected with pSpCas9(BB)-2A-GFP with sgRNA targeting WIPI4 exon 3 or its control construct were cultured for 48 h before mitochondrial purification and lipid extraction (after calibration to protein concentrations). f,g, Levels of PE in mitochondria (f) and whole-cell lysates (g) of ATG2A/B-KO HeLa cells transfected with GFP, GFP-ATG2A-WT or GFP-ATG2A-LTD (n = 3). h, LDH release from ATG2A/B-KO HeLa cells transfected with GFP, GFP-ATG2A-WT or GFP-ATG2A-LTD (n = 7). i, Representative bright-field (left) and fluorescence (right) images of the phenotypic abnormalities found in clutches injected with 200 pg GFP empty vector or GFP-ATG2A-WT constructs compared with their uninjected siblings at 24 h post fertilization (h.p.f.). Injection of GFP-tagged constructs resulted in green-fluorescent fish with a mosaic pattern. Higher levels of toxicity were observed for the clutches injected with GFP-ATG2A-WT compared with the GFP-injected clutches and their uninjected siblings, as indicated by more dead embryos (eggs containing black dense material) or abnormal phenotypes (yellow arrowheads). The higher-magnification images (right) showcase the range of morphological defects observed. Scale bars, 2 mm (main images showing clutches) and 1 mm (higher-magnification images showing individual embryos). j, Day 15 i³ neurons were pre-treated with DMSO (control), 10 µM Z-VAD-fmk (Z-VAD), 20 µM necrostatin (Nec-1), 500 nM liproxstatin-1 (Lip-1), 5 µM Fer-1, 10 µM MitoTEMPO (MT), 10 nM MitoQH₂ (QH₂) or 5 µM inPISD for 12 h, followed by scramble shRNA or shWIPI4 lentivirus treatment in combination with the same drugs for 24 h. LDH release was then assayed as a measure of cytotoxicity (n = 3). a–g, Data are the mean ± s.d.; two-tailed paired Student’s t-test. h,j, Data are the normalized mean ± s.d.; two-tailed one-sample Student’s t-test for comparisons with scramble DMSO and two-tailed paired Student’s t-test for comparisons with other samples. a–h,j, n, number of biologically independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001. Source numerical data are provided.

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ATG2 transports PS into mitochondria, which is then locally converted to PE.

a, HeLa cells were transfected with scramble siRNA, siWIPI4 or siRNA targeting mitochondrial PISD-2 (simPISD) for 48 h and the levels of LDH release were measured (n = 3). b, ATG2A/B-KO HeLa cells were transfected with scramble siRNA or simPISD for 48 h before transfection with GFP or GFP–ATG2A constructs for another 24 h. Conditioned media were used for the LDH assay (n = 3). c,d, Cells from b were lysed and assayed for MDA (c) and PE (d). Two-tailed paired Student’s t-test (n = 3). e, Cell death of HeLa cells transfected with CMV-myc-DDK or mitoPISD-myc-DDK (n = 3). The cells were cultured for 24 h before treatment with DMSO or 100 nM Lip-1 in media containing IncuCyte red dye. The samples were imaged 48 h post transfection and dead cells were counted (number of red objects). Data are the average number of dead cells per well normalized to the CMV-myc-DDK-transfected/DMSO-treated control. f, Mitochondrial conversion of 18:1-12:0 NBD-PS to PE within 1.5 h measured by microscopy (n = 3). g, HeLa cells were first treated with Seahorse for 15 min and then treated, imaged and assayed as in f (n = 4). h, Cell death of SH-SY5Y cells transfected with 50 nM siWIPI4 and/or siRNA to ORP5/8 (siORP5/8; n = 3). Cells were stained with CellTox green dye 48 h post transfection. Cell death was determined as the ratio of the green area (dead cells) to the phase area (total cells). i, Levels of PE in the mitochondrial fractions of HeLa cells transfected with the indicated siRNAs (n = 3). Cell fractionation was performed 48 h post transfection and mitochondrial samples with same protein abundance were used for the PE assay. a,b,e,h, Data are the normalized mean ± s.d. Two-tailed one-sample Student’s t-test for comparisons to the siWIPI4 (a) or controls (b,e,h) and two-tailed paired Student’s t-test for comparisons between other samples. f,g,i, Data are the mean ± s.d. Two-tailed paired Student’s t-test. a–i, n, number of biologically independent experiments. *P < 0.05, **P < 0.01 and ***P < 0.001. Source numerical data are provided.

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