Figure 5.
Figure 5. Depletion of CHMP6 and overexpression of dominant-negative CHMP6 results in abscission delay. (A) Cumulative frequency plot showing the time interval between furrow ingression and abscission in HeLa cells stably expressing Histone2B-mCherry, eGFP–α-tubulin, and VPS28 alleles upon siRNA treatment as indicated (n ≥ 100 cells per treatment from four independent experiments; control, 61 ± 15.2 min; VPS28 siRNA, 73 ± 15.0 min; VPS28 with VPS28 siRNA, 58 ± 14.0 min; VPS28mut with VPS28 siRNA, 88 ± 18.1 min; VPS28ΔCTD with VPS28 siRNA, 86 ± 23.3 min [± SD]; control and VPS28 rescue relative to VPS28 siRNA, P < 0.01; control and VPS28 rescue relative to VPS28mut and VPS28ΔCTD, P < 0.01; VPS28 siRNA relative to VPS28mut and VPS28ΔCTD, P < 0.01; P-values obtained using mixed factor model). (B) Knockdown efficiency of VPS28 and codepletion of TSG101. (C) Cumulative frequency plot showing the time interval between furrow ingression and abscission in HeLa cells stably expressing Histone2B-mCherry and eGFP–α-tubulin upon siRNA treatment as indicated (n ≥ 200 cells per treatment from seven independent experiments; control, 69 ± 14.9 min; CHMP6 siRNA#1, 104 ± 53.7 min; CHMP6 siRNA#2, 85 ± 28.2 min [± SD]; CHMP6 1 relative to control, P < 0.001; CHMP6 2 relative to control, P < 0.001; P-values obtained using mixed factor model). (D) Knockdown efficiency of CHMP6. (E) Schematic view of the CHMP6 constructs used in F and G. (F) Confocal images of HeLa cells transfected with wild-type or truncated CHMP6 and stained for DNA (Hoechst), α-tubulin, and MKLP1. Bars, 5 µm. CHMP6core localizes to the midbody. (G) HeLa cells were transfected with wild-type or truncated CHMP6 and analyzed by high-throughput widefield microscopy. The mean of the total cell population is shown (error bars indicate SEM from six independent experiments; n > 1,000 cells per experiment; unpaired t test; ***, P < 0.001).

Depletion of CHMP6 and overexpression of dominant-negative CHMP6 results in abscission delay. (A) Cumulative frequency plot showing the time interval between furrow ingression and abscission in HeLa cells stably expressing Histone2B-mCherry, eGFP–α-tubulin, and VPS28 alleles upon siRNA treatment as indicated (n ≥ 100 cells per treatment from four independent experiments; control, 61 ± 15.2 min; VPS28 siRNA, 73 ± 15.0 min; VPS28 with VPS28 siRNA, 58 ± 14.0 min; VPS28mut with VPS28 siRNA, 88 ± 18.1 min; VPS28ΔCTD with VPS28 siRNA, 86 ± 23.3 min [± SD]; control and VPS28 rescue relative to VPS28 siRNA, P < 0.01; control and VPS28 rescue relative to VPS28mut and VPS28ΔCTD, P < 0.01; VPS28 siRNA relative to VPS28mut and VPS28ΔCTD, P < 0.01; P-values obtained using mixed factor model). (B) Knockdown efficiency of VPS28 and codepletion of TSG101. (C) Cumulative frequency plot showing the time interval between furrow ingression and abscission in HeLa cells stably expressing Histone2B-mCherry and eGFP–α-tubulin upon siRNA treatment as indicated (n ≥ 200 cells per treatment from seven independent experiments; control, 69 ± 14.9 min; CHMP6 siRNA#1, 104 ± 53.7 min; CHMP6 siRNA#2, 85 ± 28.2 min [± SD]; CHMP6 1 relative to control, P < 0.001; CHMP6 2 relative to control, P < 0.001; P-values obtained using mixed factor model). (D) Knockdown efficiency of CHMP6. (E) Schematic view of the CHMP6 constructs used in F and G. (F) Confocal images of HeLa cells transfected with wild-type or truncated CHMP6 and stained for DNA (Hoechst), α-tubulin, and MKLP1. Bars, 5 µm. CHMP6core localizes to the midbody. (G) HeLa cells were transfected with wild-type or truncated CHMP6 and analyzed by high-throughput widefield microscopy. The mean of the total cell population is shown (error bars indicate SEM from six independent experiments; n > 1,000 cells per experiment; unpaired t test; ***, P < 0.001).

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