Fig. 4: Membrane damage correlates with CHMP4B activation.

Time-lapse live confocal images of NIH-3T3 cells transiently transfected with A CHMP4B-mCherry or B CHMP4B-eGFP, treated with RSL3, and monitored for CHMP4B puncta formation, Fluo-4 AM, and PI staining. Scale bar, 20 µm. C, D Kinetics of the appearance of CHMP4B puncta and increase of cytosolic Fluo-4 AM signal, change in cell rounding, and PI intake upon RSL3 treatment. Plots show the average temporal relationships between normalized C mCherry (N = 8 cells) or D eGFP (N = 15 cells) fluorescence intensity standard deviation and cell rounding. All cells were synchronized to the first appearance of CHMP4B puncta (t = 0). Interval time between measurements was 5 min. E Time delay between the appearance of CHMP4B puncta, Ca²⁺ signal, cell rounding, and PI. t₅₀ of each event was calculated from individual curves obtained per single cells (shown in C and D). These values correspond to the time at 50% of the maximum signal and were plotted as lag time with respect to appearance of the CHMP4B puncta. F Graphical representation of the sequence of events related to CHMP4B activation in ferroptosis. G Time-lapse images of NIH-3T3 cells transiently transfected with CHMP4B-mCherry and treated with RSL3 (2 µM) in the presence of PEG 8000. Scale bar, 15 µm. H Kinetics of the appearance of CHMP4B puncta and change in cell rounding upon RSL3 treatment in the presence or absence of PEG 8000. Plots show the average temporal relationships between normalized cell rounding and the folding increase of the standard deviation of mCherry (n = 8 cells in the experiment with PEG 8000 and 6 cells in the experiment without PEG 8000). The activation of ESCRT-III was measuring as the fold increase of the SD of the mCherry CHMP4B fluorescence signal over time. Interval time between measurements was 5 min. I Fold increase of the Fluo-4 AM mean intensity and percentage of cells containing CHMPH4 puncta in the presence or absence of PEG 8000.