Extended Data Fig. 2: The phospholipid peroxidation sensor Bodipy C11 colocalizes primarily with EMCSs and subsequently with the mitochondria.
a, Representative 2D images of MEFs transiently transfected with the ER marker BFP-KDEL (cyan) and stained with MitoTracker Far Red (magenta) and Bodipy C11 (green) at time 0 and at 5’, 15’, 30’ after RSL3 (0.5 µM). EMCS masked area, representing colocalization of the ER and mitochondria, is indicated as white. Magnified images represent respectively ox-Bodipy together with i) ER surface, ii) EMCS mask and iii) mitochondrial surfaces together with EMCS mask. Scale bar, 10 µm. Zoom, 1 µm. b-e, Colocalization (% M1, Mander’s coefficient) of oxidized Bodipy C11 (ox-Bodipy C11) (green) signal in the ER, EMCS mask, mitochondria and the residual cellular region outside the ER + mitochondrial masks, at time 0 and 5, 15, 30‘ after RSL3 (0.5 µM). (n = 3 biological replicates). f, Total Cell Liperfluo intensity normalized on total cellular volume (MFI) at time 0 and 5, 15, 30‘ after RSL3 (0.5 µM). (n = 3 biological replicates). g, Total Cell ox-Bodipy C11 intensity (green) normalized on non-oxidized Bodipy C11 (red) at time 0 and 5, 15, 30‘ after RSL3 (0.5 µM). (n = 3 biological replicates). All quantitative data are mean ± SEM. In b-e, g, statistical significance was determined by one-way ANOVA, Dunnett post-hoc test. In f, statistical significance was determined by RM one-way ANOVA, Dunnett 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.
