Fig. 1

Fig. 1 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.

Source data