Fig. 6

Fig. 6 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.

Source data