Figure 5. VPS4A Suppression Leads to ESCRT-III Filament Accumulation, Deformed Nuclei, and Abscission Defects in VPS4B^loss Cancer Cells.

(A) Known functions of the ESCRT machinery in membrane biology.

(B) Digitized immunoblot showing VPS4A and Vinculin protein levels in VPS4B^neutral and VPS4B^loss cancer cell lines with the dox-inducible shVPS4A-2 RNAi system after 5 days of treatment with control or 1 μM dox.

(C) Confocal immunofluorescence of CHMP4B in four cancer cell lines with the dox-inducible shVPS4A-2 RNAi system, from (B) imaged after 6 days of treatment. (grayscale, scale bars: 50 μm).

(D) Quantification of CHMP4B speckle formation in untreated (orange) and dox-treated (blue) cells from (C) on multiple random images (n = 3–9). ns, not significant; **q < 0.01, ****q < 0.0001.

(E) Confocal fluorescence imaging of DNA (DAPI, blue) of parental RD-SpCas9 cancer cells euploid for VPS4B copy and clone B2 RD-SpCas9 cancer cells with knockout of VPS4B (Figures 4F, 4G, and S6D). Scale bars: 50 mm. Nuclear surface size by CellProfiler. ****p < 0.0001.

(F) Confocal (immuno)fluorescence of the inner nuclear membrane protein Emerin (Alexa Fluor 561, red) and DNA (DAPI, blue) in four different cancer cell lines, from (B) after 6 days of treatment. Arrows: micronuclei. Scale bars: 50 μm.

(G) (Immuno)fluorescence of cytokinetic bridges and midbodies using tubulin (Alexa Fluor 488, green) and DNA (DAPI) in 3 different cancer cell lines after 4 days of induction of CRISPR-SpCas9-mediated disruption of an intergenic region (sgChr2–2) or VPS4A (sgVPS4A-1). Arrows: cytokinetic bridges.

(H) Quantification of cancer cells connected to neighboring cells by cytokinetic bridges. ns, not significant; **p < 0.01, ****p < 0.0001.

See also Figure S7.