Fig. 4: ALIX, syntenin, and syndecan-4 are required for successful abscission.

a Transduced-HeLa cell lines expressing either GFP, wild-type ALIX-GFP or ALIX F676D-GFP were treated with either control or ALIX siRNAs. Abscission time (from furrow onset to abscission) was determined by phase-contrast time-lapse microscopy. Cumulative plot of the abscission times (upper panels) and mean abscission times (means ± SD) (lower panels) are shown. n = 111–156 cells, N = 3 independent experiments. b Abscission time was determined as above in transduced cell lines expressing either GFP, wild-type mCherry-syntenin, mCherry-syntenin ΔALIX, or mCherry-syntenin ΔSDC and treated with either control or syntenin siRNAs, as indicated. n = 105–147 cells, N = 3 independent experiments. c Abscission time was determined as above in HeLa cells treated with either control or syndecan-4 siRNAs, and transfected with either empty plasmid or plasmid expressing siRNA resistant transcript encoding syndecan-4. n = 130–179 cells, N = 3 independent experiments. Time axis were stopped at 1200 min. d Western blots of protein extracts from cells treated with control, ALIX, syntenin (Synt), syndecan-4 (SDC4), TSG101 siRNAs, or a combination of these siRNAs and revealed with the indicated antibodies. Loading control: calreticulin. e–g Abscission time was determined in cells treated with either control, ALIX, syntenin, syndecan-4, TSG101 siRNAs, or a combination of these siRNAs. Mean abscission times (means ± SD) are depicted in (h). n = 120–152 cells, N = 3 independent experiments. All measurements were carried out in parallel but have been displayed in three graphs for clarity. Upper panels a–c, e–g (time distribution): nonparametric and distribution-free Kolmogorov–Smirnov KS tests. NS nonsignificant. Lower panels a–c, h (mean abscission times): one-sided Student’s t tests.