ALIX, but not TSG101, controls furrow regression. (A) Live imaging of HeLa cells stably expressing Histone2B-mCherry and eGFP–α-tubulin upon siRNA treatment as indicated. Percentage of cells undergoing furrow regression (error bars indicate SEM; n > 90 cells from three independent experiments; unpaired t test; **, P < 0.01; n.s., not significant). (B) Live imaging of HeLa cells stably expressing mCherry-CEP55 and CHMP4B-eGFP upon siRNA treatment as indicated. Selected frames of ALIX-depleted cells undergoing cytokinesis, showing enrichment of CHMP4B at the midbody at time of furrow regression (top, merged channels; bottom, CHMP4B-eGFP channel alone. Bar, 5 µm. (C) Confocal images of HeLa cells transfected with indicated siRNAs and stained for DNA (Hoechst), α-tubulin, RacGAP1, GFP, CHMP3, and IST1, as indicated. Bars, 5 µm. (left) HeLa cells stably expressing CHMP4B-eGFP. (D) Localization of CHMP4B to the secondary ingression of HeLa cells is unaffected by ALIX knockdown. Bars, 3 µm.