The effect of puromycin treatment on CHMP4B targeting to the MB in synchronized cells. (A) Synchronization and puromycin treatment schematics. (B and C) Synchronized HeLa cells were incubated for 90 min in the presence or absence of puromycin (during last 30 min of incubation). Cells were then foxed and stained with anti-CHMP4B antibodies (red). Asterisk marks the midbody. Scale bars: 10 μm. C shows quantification of CHMP4B localization in the midbody. Data shown are the means and SD calculated from individual cell values. Dots represent localization in individual cells. (D) HeLa cells were either untreated, treated with puromycin, CEP55 siRNA, or CEP55 siRNA plus puromycin and subjected to immunostaining anti-acetylated-tubulin and anti-CHMP4B antibodies. The asterisks mark the MB, and the white dashed square represents the region of the image used for the inset. Scale bars: 10 μm. Inset scale bars: 2 μm. (E and F) Shown data represents CHMP4B intensity/μm² in intercellular of 60 cells from three separate experiments. E shows distributions derived from individual cells (each dot represents a single cell). Statistical analysis in F was done on the means and SD were derived from the three individual experiments where experimental means were calculated by averaging values from all the cells from each experiment. Each experiment is represented by a different shade of color. Statistical analysis is represented with a P value.