Fig. 4: Spacing EMCSs protects cells from EMCS-mediated mitochondrial PLox.
a, A schematic representation of 9xL spacer. b, Representative volume-rendered 3D reconstructions of confocal z-stacks from time-lapse live images of MEFs transiently transfected with AKAP1–mRFP control or 9xL spacer and the ER marker BFP–KDEL (cyan) and stained with MitoTracker Far Red (magenta) and Liperfluo (green) at time 0, 5, 15 and 30 min after RSL3 (0.5 µM). The EMCS masked volume, representing colocalization of the ER and mitochondria, is indicated in white. The magnified images represent, respectively, (i) the merge of ER, mitochondrial surfaces and EMCS mask and (ii) Liperfluo and mitochondrial surfaces together with EMCS mask. Scale bar, 10 µm. Zoom, 1 µm. c, A quantification of the per cent of EMCSs normalized on the ER volume (BFP–KDEL) at time 0, 5, 15 and 30 min after RSL3 (0.5 µM) in MEFs transiently transfected with AKAP1–mRFP control or 9xL spacer (n = 3 biological replicates). ctrl, control. d–f, A colocalization (Mander’s coefficient (M1), per cent) of Liperfluo in the EMCS (d), mitochondria (e) and ER (f) masks at time 0, 5, 15 and 30 min after RSL3 (0.5 µM) treatment in MEFs transiently transfected with AKAP1–mRFP control or 9xL spacer (n = 3 biological replicates, the same cells (n = 7 mRFP control, n = 7 9xL spacer) were acquired and analysed at each timepoint per condition). Similar experiments and settings have been reproduced independently on Fig. 2, Supplementary Fig. 2 and Supplementary Fig. 3. All quantitative data are the mean ± s.e.m. In c, d, e and f, the statistical significance was determined by a two-way repeated-measures ANOVA, Tukey post hoc test. n.s., not significant (P > 0.05), *P ≤ 0.05, **P < 0.01, ***P < 0.001. Panel a created with BioRender.com.
