Fig. 6: ER–mitochondria tethering fosters ferroptosis.

a, The per cent of cell death of PERK^WT and PERK^KO MEFs at time 0 and after RSL3 (0.125 µM, 24 h) in the absence or presence of ZVAD (30 µM), Nec1s (30 µM), DFO (50 µM) or Fer-1 (1 µM) (n = 4 biological replicates, three wells per condition per biological experiment). b, Representative images of live cells from PERK^WT, PERK^KO and PERK^KO MEFs after transient re-expression of PERK^KD at time 0 (T0) and 24 h (T24h) after RSL3 (0.5 µM). The images show SYTOX Green staining and bright field (BF). Scale bar, 100 µm. c, The per cent of cell death in PERK^WT, PERK^KO and PERK^KO MEFs after transient re-expression of PERK^KD after RSL3 (0.5 µM, 24 h) (n = 3 biological replicates). d, Representative images of live cells from MFN2^KO MEFs transiently transfected with HA-tag-EV, MFN2-HA and ERMIT2-HA at T0 and after RSL3 (0.5 µM, 24 h). The images show SYTOX Green staining and BF. Scale bar, 100 µm. e, The per cent of cell death in MFN2^KO MEFs transiently transfected with HA-tag-EV, MFN2-HA and ERMIT2-HA after RSL3 (0.5 µM, 24 h). Scale bar, 100 µm (n = 3 biological replicates). f, Representative images of live cells from PERK^WT MEFs transiently transfected with AKAP1–mRFP control or 9xL spacer at time 0 and 24 h after RSL3 (0.5 µM). The images show SYTOX Green staining, BF and RFP. Scale bar, 100 µm. g, The per cent of cell death in PERK^WT MEFs transiently transfected with AKAP1–mRFP control or 9xL spacer after RSL3 (0.5 µM, 8 h) (n = 3 biological replicates). h, A schema of the OMM–ER linker. i, Representative images of live cells from PERK^WT and PERK^KO MEFs transiently transfected with AKAP1–mRFP control and PERK^KO transiently transfected with the OMM–ER linker at time 0 and 24 h after RSL3 (0.5 µM). The images show SYTOX Green staining, BF and RFP. Scale bar, 100 µm. j, The per cent of cell death of PERK^WT and PERK^KO MEFs transiently transfected with AKAP1–mRFP control and PERK^KO transiently transfected with the OMM–ER linker after RSL3 (0.5 µM, 24 h). Scale bar, 100 µm (n = 3 biological replicates). All the quantitative data are the mean ± s.e.m. In a, the statistical significance was determined by a two-way ANOVA, Sidak post hoc test. In c, e and j, the statistical significance was determined by a one-way ANOVA, Tukey post hoc test. In g, the statistical significance was determined by a two-sided unpaired t-test. n.s., not significant (P > 0.05), *P ≤ 0.05, **P < 0.01, ***P < 0.001.

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