Figure 6.

Knockdown of rhotekin impairs cell proliferation by cytokinectic delay and failure. (A) HeLa cells infected with a lentivirus expressing control (siCtrl) or shRNAs against human rhotekin (siRTKN-1, siRTKN-2) were harvested for immunoblotting of rhotekin and β-actin. (B) The proliferation rate of siCtrl and siRTKN cells monitored by Prestoblue assay. (C) Double thymidine-arrested siCtrl and siRTKN cells were released to fresh medium for 12 h, then underwent α-tubulin immunostaining and Hoechst labeling. The midbody stage is defined when 2 daughter cells are still connected by an intercellular bridge (arrows). Scale, 50 μm. The distribution of siCtrl or siVxn cells at the midbody stage and interphase is expressed as relative ratios with the ratio in siCtrl cells arbitrarily set to 1. Total cell number for each siRNA group from 3 independent experiments is ∼1000. (D) Time-lapse comparison of cytokinesis in a representative siCtrl cell (top) and siRTKN cell (middle). G1/S-synchronized siCtrl and siRTKN cells after release from the thymidine block were time-lapse recorded. Midbodies remained at the center of intercellular bridge and midbody remnants inherited by a daughter cell after abscission are denoted by arrows and arrowheads, respectively. Some siRTKN cells underwent apoptosis (bottom). The first image of a cell rounding up was considered time 0. (E) For recorded cells with complete cytokinesis, the time required to complete cell abscission is expressed as mean ± SEM. (F) The percentage of recorded siCtrl and siVxn cells with incomplete cytokinesis eventually forming a bi-nucleated cell. The cell numbers in each group used for analysis are labeled inside the bars. * P < 0.05 by Student t test.