Fig. 5

Fig. 5 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.