Fig. 5: SMYD2 accelerates abscission timing when cytokinesis is challenged.
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 SMYD2_GFP treated with control or Nup153 siRNAs (N = 3). Loading control: Ponceau red. B Left panels: representative cell images. Yellow arrows show cytokinetic cells connected by an ICB. Scale bar = 10 um. Right panel: quantification of cytokinetic cells (N = 3, n > 300 cells, mean ± SD, Two-sided multiple unpaired t-test, ns non-significant (p > 0.05)). C Left panels: frames of the indicated HeLa cell lines. Time 00 is set prior the ICB formation. Last time point corresponds to the ICB cut, indicated by the arrow. Scale bar = 10 um. Right panel: distribution of the abscission time measured by phase-contrast time-lapse microscopy (N = 3, n > 126 cells, Two-sided Kolmogorov–Smirnov test, ns non-significant (p > 0.05)) and mean abscission duration (N = 3, mean ± SD, t-test, ns non-significant (p > 0.05)) are shown. D Quantification of ICBs with either no CHMP2B, CHMP2B in rings at the midbody, and CHMP2B both at the midbody and at the abscission site (N = 5, n > 40 ICBs counted/N, mean ± SD, Two-sided multiple unpaired t-test, ns non-significant (p > 0.05)). E Quantification of ICBs with either no VPS4A, VPS4A at midbody rings, and VPS4A both at the midbody and at the abscission site (N = 3, n > 50 ICBs counted/N, mean ± SD, Two-sided multiple unpaired t-test, ns non-significant (p > 0.05)). F Left panels: selected phase contrast time-lapse and fluorescent videomicroscopy frames of HeLa cell lines with Lap2_ β_RFP that stains for chromatin bridges (indicated by 3 arrows). Last time point corresponds to the ICB cut. Scale bar = 10 um. Right panel: distribution of the abscission time measured by phase-contrast time-lapse microscopy plotted as individual points. (n = 74, n = 18, for GFP cells with or without chromatin bridge and n = 53; n = 62 for GFP-SMYD2 cells with or without chromatin bridge, N = 3 experiments, mean ± SD, Two-sided Mann–Whitney test, ns non-significant (p > 0.05)). G Left panels: selected frames of HeLa cell lines treated with Rab35 siRNA. Time 00 is set prior the ICB formation. Last time point corresponds to the ICB cut. Scale bar = 10 um. Right panel: distribution of the abscission time measured by phase-contrast time-lapse microscopy (N = 3, n > 166 cells, Two-sided Kolmogorov–Smirnov test, ns non-significant (p > 0.05)) and mean abscission duration (N = 3, mean ± SD, t-test, ns non-significant (p > 0.05)).
