Extended Data Fig. 4: WIPI4–ATG2 axis is a novel ferroptosis pathway.
From: Loss of WIPI4 in neurodegeneration causes autophagy-independent ferroptosis
a. Cell viability was measured by Aquabluer staining in ATG2A/B WT and KO HeLa cells treated with DMSO, 10 µM erastin, 3 µM RSL3, 1 µM iFSP1, 100 nm Lip-1, or indicated combinations of these drugs (n = 3). b. Cell cytotoxicity was examined by LDH assay in ATG2A/B WT and KO HeLa cells treated with 3 µM RSL3, 10 µm sorafenib, 10 µM erastin,5 µM FIN56 with or without 100 nm Lip-1 for 24 hours (n = 3). P values could be found in the numerical source data. c. Cell viability measured by Aquabluer assay in ATG2A/B WT and KO HeLa cells treated with 1.25 µM, 2.5 µM, or 5 µM arachidonic acid or same concentration of BSA, with or without 100 nM Lip-1 (n = 5) for 24 hours. d. HeLa cells transfected with control or WIPI4 siRNAs for 72 hours were treated with 1 µM Fer-1 for 24 hours. The cells were incubated with Bodipy581/591C11 for 15 minutes before cell lysis and were then subjected to needle homogenization without detergent prior to immunoprecipitation with endogenous ATG2A. Indicated fluorescent strength on the objects attached to magnetic beads was observed with confocal microscopy. This experiment was done twice with HeLa and SH-SY5Y cells. e. Quantification of representative experiments measuring the shift of Bodipy C11 signal on ATG2A pulled-down beads in SH-SY5Y cells (upper, n = 1) and HeLa cells (lower, n = 1). Datapoints represent the ratio of two fluorescent levels (510 nm, oxidized / 590 nm, reduced) of each taken field. Median with 95% CI. Data and error bars in a-c are normalized mean ± SD. Two-tailed one sample t-test to control and two-tailed paired t-test between other samples. N represents the number of biologically independent experiments. Source numerical data are available in source data.
