Fig. 3: CHMP2B K6 is involved in the timely progression of CHMP2B to the abscission site.
From: Methylation of ESCRT-III components regulates the timing of cytokinetic abscission
A Western blots of protein extracts from HeLa cell lines stably expressing GFP or siRNA resistant (siR) CHMP2B_GFP or CHMP2B K6A_GFP treated with control or CHMP2B siRNAs revealed with CHMP2B antibody (N = 3, CHMP2B exo. = CHMP2B_GFP protein, CHMP2B endo. = endogenous CHMP2B). Loading control: Ponceau red staining. B Selected frames from phase-contrast time-lapse videomicroscopy of HeLa cells stably expressing GFP treated with control or CHMP2B siRNAs entering in cytokinesis and completing their abscission. Time is expressed in minutes, and time 00 is set at the time point prior the intercellular bridge formation. Last time point corresponds to the ICB cut indicated by the empty arrow, considered here as the abscission (N = 3). Scale bar = 10 um. C Distribution of the abscission time measured by phase-contrast time-lapse microscopy in the indicated cells (N = 3, n > 109 cells, Two-sided Kolmogorov–Smirnov test, ns non-significant (p > 0.05)) and the mean abscission duration (minutes, N = 3, mean ± SD, t-test, ns non-significant (p > 0.05)) are shown. D Quantification of the abscission time of HeLa cell lines stably expressing CHMP2B_GFP or CHMP2B K6A_GFP (from furrow onset to the microtubule cut), each dot represents one dividing cells. (N = 3, n > 50, median (red lines) and quartiles (dotty lines), Two-sided Mann–Whitney test). E Left panel: live cell imaging of HeLa cell lines stably expressing CHMP2B_GFP (top) or CHMP2B K6A_GFP (bottom) treated with siR-tubulin (magenta). Time is expressed in minutes, and time 00 is set at the time point prior the intercellular bridge formation. The last time point corresponds to the microtubule cut, considered here as the abscission. Scale bar = 5 um. Right panel: duration of each abscission stage measured from live cell imaging performed in CHMP2B_GFP or CHMP2B K6A_GFP expressing cells (N = 3, n > 50, median (red lines) and quartiles (dotty lines), Two-sided Mann–Whitney test, ns non-significant (p > 0.05)).
