CHMP4C recruitment to the midbody relies on ALIX. (A) CHMP4A recruitment is maintained in the absence of ALIX. Confocal images of HeLa cells upon siRNA treatment as indicated, stained for DNA (Hoechst), CHMP4A, α-tubulin, and RacGAP1. Bars, 5 µm. (B) CHMP4C recruitment to the midbody is perturbed in the absence of ALIX. Live imaging of HeLa cells stably expressing inducible eGFP-CHMP4C upon siRNA treatment as indicated. Images and quantification of the relative intensity of eGFP-CHMP4C at the midbody are shown (bars indicate mean with 95% confidence interval; n ≥ 30 cells from four independent experiments; mixed factor model; ***, P < 0.001). Bars, 5 µm. (C) Schematic view of the CHMP4C constructs used in D–F. (D) Confocal images of HeLa cells stably expressing CHMP4C-V5 or CHMP4C^ΔALIX-V5, stained for DNA (Hoechst), V5, α-tubulin, and MKLP1. Bars, 5 µm. (E) Quantification of full-length or truncated CHMP4C-V5 at the midbody (error bars indicate SEM; n > 50 cells from six independent experiments; unpaired t test; ***, P < 0.001). (F) Depletion of ALIX increases frequency of binucleation in the presence of lagging chromosomes. Live imaging of HeLa cells stably expressing Histone2B-mCherry and eGFP-α-tubulin upon siRNA treatment as indicated. Quantification of furrow regression in cells with lagging chromosomes as depicted in the cartoon (error bars indicate SEM; n = 24 cells from three independent experiments; unpaired t test; *, P < 0.05). (G) Live imaging of HeLa cells stably expressing Histone2B-mCherry, eGFP-α-tubulin, and siRNA-resistant CHMP4C or CHMP4C^ΔALIX upon CHMP4C siRNA treatment. Quantification of furrow regression in cells with lagging chromosomes (error bars indicate SEM; n ≥ 15 cells from three independent experiments; unpaired t test; *, P < 0.05).