Extended Data Fig. 9: PERK kinase activity is dispensable for promoting ferroptosis.

a, Dose response of RSL3 induced cell death (% Sytoxgreen positive cells) of PERK^WT and PERK^KO MEFs 24 h after treatment. (n = 3 biological replicates). b, Dose response of IKE induced cell death (% Sytoxgreen positive cells) of PERK^WT and PERK^KO MEFs 24 h after treatment. (n = 3 biological replicates). c, Time course of RSL3 (0.125 µM) induced cell death (% Sytoxgreen positive cells) of PERK^WT and PERK^KO MEFs. (n = 3 biological replicates). d, Time course of IKE (0.625 µM) induced cell death (% Sytoxgreen positive cells) of PERK^WT and PERK^KO MEFs. (n = 3 biological replicates). e, % cell death (Sytoxgreen positive cells) of PERK^WT and PERK^KO MEFs treated for 24 h with IKE (0.625 µM) treatment, in the absence or presence of Z-Val-Ala-DL-Asp(OMe)-fluoromethylketone (ZVAD, 30 µM), Necrostatin-1s (Nec1s, 30 µM), deferoxamine mesylate salt (DFO, 50 µM) or ferrostatin 1 (Fer-1, 1 µM).(n = 3 biological replicates) f, Representative immunoblot for PERK in PERK^WT and PERK^KO MEFs and PERK^KO transiently re-expressing PERK^KD cells. ACTIN serves as a loading control. g, % cell death (Sytoxgreen positive cells) of PERK^WT and PERK^WT MEFs in the absence or presence of the GSK2606414 PERK inhibitor (PKI), treated for 24 h with RSL3 (0.125 µM) in the absence or presence of Z-Val-Ala-DL-Asp(OMe)-fluoromethylketone (ZVAD, 30 µM), Necrostatin-1s (Nec1s, 30 µM), deferoxamine mesylate salt (DFO, 50 µM) or ferrostatin 1 (Fer-1, 1 µM). (n = 3 biological replicates). h, % cell death (Sytoxgreen positive cells) of eIF2α (S/S) and eIF2α (S/A) MEFs treated for 24 h with RSL3 (0.125 µM), in the absence or in the presence of Fer-1 (1 µM). (n = 3 biological replicates). i, Representative immunoblot for HA and MFN2 in MFN2^WT and MFN2^KO MEFs transiently re-expressing HA, MFN2-HA and ERMIT2-HA cells. ACTIN serves as a loading control. j, Representative FRET live microscopy images of PERK^KO MEFs transiently transfected with AKAP1-mRFP control or OMM-ER linker and transiently infected with FEMP probe (mAKAP1-YFP-Tav2-CFP-Sac1). Scale bar 10 µm. Zoom, 10 µm. k, FEMP ratio in PERK^KO MEFs transiently transfected with AKAP1-mRFP control or OMM-ER linker and transiently infected with FEMP probe. (n = 3 biological replicates). l, Representative images of live PERK^WT cells transiently transfected with empty vector (EV) GFP or S1R-GFP at T0 and 8 h after RSL3 (0.5 µM). Images were taken for GFP, PI and bright-field (BF). Scale bar, 100 µm. m, % cell death (PI positive cells/total cell number) of PERK^WT MEFs transiently transfected with empty vector (EV) GFP or S1R-GFP 8 h after RSL3 (0.5 µM). (n = 3 biological replicates). n, Representative images of live PERK^WT and PERK^KO cells transiently transfected with empty vector (EV) GFP and PERK^KO cells transiently transfected with S1R-GFP at T0 and 24 h after RSL3 (0.5 µM) treatment. Images were taken for GFP, PI, and bright-field (BF). Scale bar, 100 µm. o, % cell death (PI positive cells/total cell number) PERK^WT and PERK^KO cells transiently transfected with empty vector (EV) GFP and PERK^KO MEFs transiently transfected with S1R-GFP after 24 h RSL3 (0.5 µM). (n = 3 biological replicates). In a, b, c, d, e, g, h 3 wells per condition per biological experiment were analyzed. In k, m, o multiple areas in the same well were acquired and analyzed at each timepoint per condition and per biological replicate. All quantitative data are mean ± SEM. In a-e, g, h statistical significance was determined by two-way ANOVA, Sidak post-hoc test. In k, m, statistical significance was determined by two sided unpaired t-test. In o, statistical significance was determined by one way ANOVA, Sidak post-hoc 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