Extended Data Fig. 6: Altering EMCS integrity protects from ferroptosis-mediated mitochondrial fragmentation.

a, Representative images of co-stained MEFs with TOMM20 and DAPI in PERK^WT and PERK^KO MEFs. Images were taken in fixed untreated cells and 30’ after RSL3 (0.125 µM). Scale bar, 10 µm. b, Mitochondrial fragmentation index in untreated cells and 30’ after RSL3 (0.125 µM) in PERK^WT and PERK ^KO MEFs. (n = 3 biological replicates). (n = 14 WT untreated, n = 12 WT 30’, n = 15 KO untreated, n = 15WT 30’) were acquired and analyzed from 3 independent biological replicates. Similar experiments and settings have been reproduced independently in Supplementary Fig. 4. c, Representative images of co-stained MEFs with TOMM20 and DAPI in PERK^WT and PERK^KO MEFs. Images were taken in fixed untreated cells and 30’ after IKE (0.625 µM). Scale bar, 10 µm. d, Mitochondrial fragmentation index in untreated cells and 30’ after IKE (0.625 µM) in PERK^WT and PERK ^KO MEFs. (n = 2 biological replicates; n = 9 WT untreated, n = 9 WT 30’, n = 9 KO untreated, n = 9 WT 30’) were acquired and analyzed from 2 independent biological replicates. Similar experiments and settings have been reproduced independently in Supplementary Fig. 4. e, Representative images of live cells of PERK^WT MEFs transiently transfected with AKAP1-mRFP control or 9xL spacer at T0 and 30’ after RSL3 (0.5 µM). Scale bar, 10 µm. f, Mitochondrial fragmentation index in MEFs transiently transfected with AKAP1-mRFP control or 9xL spacer in untreated cells and 30’ after RSL3 (0.5 µM). (n = 3 biological replicates). g, % oxidized MitoPerox (ox-MitoPerox) in PERK^WT and PERK^KO MEFs 3 h after RSL3 (0.125 µM) measured by FACS as a shift in MitoPerox fluorescence and relative to PERK^WT. (n = 4 biological replicates). h, % oxidized Bodipy C11 (ox-Bodipy C11) in PERK^WT and PERK^KO MEFs 6 h after RSL3 treatment measured by FACS as a shift in Bodipy C11 red/green fluorescence and relative to PERK^WT. (n = 3 biological replicates). All quantitative data are mean ± SEM. In b, d, statistical significance was determined by two-way ANOVA, Tukey post-hoc test. In f, statistical significance was determined by two-way RM ANOVA, Sidak post-hoc test. In g, h, statistical significance was determined by one sample t-test. NS, not significant (P > 0.05), *P ≤ 0.05, **P < 0.01, ***P < 0.001. Source numerical and statistical data are provided.

Source data