CHMP6 and ESCRT-II localize to the cytokinetic bridge and contribute to CHMP4B recruitment. (A and C) Confocal images of HeLa cells stained for DNA (Hoechst), α-tubulin, CHMP6, and EAP20 upon siRNA treatment as indicated. Bars, 5 µm. (B and D) Intensity of CHMP6, EAP20, and α-tubulin along the intercellular bridge (error bars indicate SEM from three independent experiments; n = 30 cells; mean CHMP6 intensity in the bridge upon CHMP6 siRNA relative to control siRNA [set to 1, ± SEM] 0.47 ± 0.07 [P = 0.02], mean EAP20 intensity upon EAP30 siRNA relative to control siRNA 0.59 ± 0.05 [P = 0.01], mean EAP20 intensity upon EAP20 siRNA relative to control siRNA 0.69 ± 0.03 [P = 0.01]; P-values obtained using one-sample t test). (E) Confocal images showing CHMP4B at the cytokinetic bridge of HeLa cells stained for DNA (Hoechst), α-tubulin, CHMP4B, and the midbody marker RacGAP1 upon siRNA treatment as indicated. Bars, 5 µm. (F) Quantification of cells with or without CHMP4B at the midbody (error bars indicate SEM; n > 30 cells from four independent experiments; unpaired t test; **, P < 0.01) upon siRNA treatment as indicated. (G) Quantification of CHMP4B recruitment to the midbody in HeLa cells stably expressing siRNA sensitive or resistant EAP30 (error bars indicate SEM; n ≥ 24 cells from three independent experiments; unpaired t test; *, P < 0.05). Knockdown of ALIX and EAP30 is shown by Western blot (*, nonspecific immunoreactivity). (H) Schematic view of cytokinetic CHMP4B recruitment.