Fig. 3: PERK deficiency protects from EMCS expansion during the early phases of ferroptosis.

a, Representative FRET live microscopy images of PERK^WT and PERK^KO MEFs transiently infected with FEMP probe (mAKAP1-YFP-Tav2-CFP-Sac1) and treated with RSL3 (0.5 µM) and PERK^WT cotreated with RSL3 (0.5 µM) + Fer-1 (1 µM) at time 0 and 15 min after RSL3 treatment. Scale bar, 10 µm. Zoom, 10 µm. b, The FEMP ratio at 0, 15 and 30 min and 1 h after RSL3 (0.5 µM) and rapamycin treatment, in PERK^WT MEFs transiently infected with a FEMP probe (n = 3 biological replicates, multiple areas in the same well were acquired and analysed per each timepoint per condition per biological replicate). MAX, maximum. c, Time-lapse imaging of the FEMP ratio traces, fold changes (on untreated) of PERK^WT and PERK^KO MEFs treated with RSL3 (0.5 µM) and PERK^WT cotreated with RSL3 (0.5 µM) + Fer-1 (1 µM) infected with the FEMP probe. Where indicated, the cells were treated with 100 nM rapamycin. d, The FEMP ratio at 0, 15 and 30 min and 1 h after RSL3 (0.5 µM) treatment and rapamycin treatment, in PERK^WT and PERK^KO MEFs and PERK^WT cotreated with RSL3 (0.5 µM) + Fer-1 (1 µM) infected with the FEMP probe (n = 3 biological replicates, multiple areas in the same well were acquired and analysed per each timepoint per condition and biological replicate). e,f, Representative images (e) of in situ PLA in PERK^WT and PERK ^KO MEFs and quantification (f) of N of dots corresponding to IP3R3–VDAC1 interaction per nucleus in untreated cells and in cells treated for 15 and 30 min and 1 h with RSL3 (0.125 µM) (n = 3 biological replicates, images n = 15 for PERK^WT untreated, n = 20 for PERK^WT 15 min of RSL3 (15′RSL3), n = 20 for PERK^WT30′RSL3, n = 19 for PERK^WT for 1 h RSL3, n = 17 for PERK^KO untreated, n = 18 for PERK^KO 15′RSL3, n = 20 for PERK^KO 30′RSL3, n = 20 for PERK^KO for 1 h RSL3 and quantified as described in Fig. 1d). Scale bar, 10 µm. All quantitative data are mean ± s.e.m. In b, the statistical significance was determined by a repeated-measures two-way ANOVA, Tukey post hoc test. In d, the statistical significance was determined by a one-sample t-test. In f, the statistical significance was determined by a two-way ANOVA, Tukey post hoc test. n.s., not significant (P > 0.05), *P ≤ 0.05, **P < 0.01, ***P < 0.001.

Source data