Extended Data Fig. 3: Time-lapse live imaging of phospholipid peroxides onset and propagation in IKE treated cells.
a, Representative volume-rendered 3D reconstructions of confocal z-stacks from time-lapse live images of MEFs transiently transfected with the ER marker Sec61ß-mCherry (cyan) and stained with MitoTracker Far Red (magenta) and Liperfluo (green) at time 0 and 5’, 15’, 30’ after IKE (0.625 µM). EMCS masked volume, representing colocalization of the ER and mitochondria, is indicated as white. Magnified images represent respectively, i) 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. b-d, Colocalization (% M1, Mander’s coefficient) of Liperfluo in the EMCS (b), mitochondria (c), ER (d) masks at time 0 and 5’, 15’, and 30’ after IKE (0.625 µM) without or with Fer-1 (1 µM) in MEFs. (n = 3 biological replicates). e, Quantification of the % EMCS normalized on the ER volume (Sec61ß-mCherry) at time 0 and 5’, 15’, 30‘ after IKE (0.625 µM) and cotreatment with Fer-1 (1 µM) in MEFs. f, Total Cell Liperfluo intensity normalized on total cellular volume (MFI) at time 0 and 5, 15, 30 ‘after IKE (0.625 µM) and cotreatment with Fer-1 (1 µM) in MEFs. (n = 3 biological replicates). (n = 3 biological replicates, the same cells (n = 11 IKE, n = 9 IKE+Fer-1) were acquired and analyzed at each timepoint per condition). Similar experiments and settings have been reproduced independently in Fig. 2, Fig. 4 and Supplementary Fig. 2. All quantitative data are mean ± SEM. In b-f, 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.
