Figure 4.

USP19 is required for successful mitotic progression

(A–E) Mock or USP19KO HCT116 cells were transfected with GFP-H2B, synchronized using thymidine and released into fresh medium. Cells were then monitored using a time-lapse microscope for 12 h and images were taken at 3-min intervals. The results were from 3 independent experiments. (A) Screenshots taken from the time-lapse microscopy of mitotic cell populations (n = 25). NEBD to the mitotic exit was estimated in the mentioned groups and graphically represented. Representative images were formed by merging GFP-H2B fluorescence and phase-contrast images. Scale bar, 25 μm. Data are presented as the means and standard deviations. A 2-tailed t test was used, and the p value is as indicated. (B) Screenshots taken from time-lapse microscopy at the indicated times from NEBD (occurred at 00:00). The anaphase onset is denoted by the red arrowhead. Yellow arrowheads show misaligned or lagging chromosomes, chromatin bridges, or improperly separated chromosomes. Scale bar, 10 μm. (C) Graphical representation of the quantification of mitotic defects from time-lapse videos (n = 10). Data are presented as the means and standard deviations of 3 independent experiments. Two-way ANOVA followed by Bonferroni post hoc test was used, and p values are as indicated. (D) Time-lapse microscopic images of cells undergoing mitosis and cytokinesis. Representative images were formed by merging GFP-H2B fluorescence and phase-contrast images. Scale bar, 25 μm. (E) Quantification of cytokinesis failure from time-lapse videos (n = 35). Data are presented as the means and standard deviations of 3 independent experiments. A 2-tailed t test was used, and the p value is as indicated. (F) Mock or USP19KO HCT116 cells were stained with TPX2 antibody (n = 5). Immunofluorescence analysis was performed to check bi- or multi-nuclei conditions and graphically represented. Scale bar, 10 μm. Data are presented as the means and standard deviations of 5 independent experiments. A 2-tailed t test was used, and the p value is as indicated. (G) Mock, USP19KO, and USP19KO cells transfected with USP19 WT or USP19CA and immunofluorescence staining was performed using TPX2-specific antibody (n = 25). HCT116 cells showing multi-spindles were counted after immunofluorescence staining and graphically represented. Data are presented as the means and standard deviations of 3 independent experiments. One-way ANOVA followed by Tukey’s post hoc test was used with the indicated p values. Scale bar, 10 μm.