PRC1 depletion reveals PLK1-independent RhoA activation and furrow ingression. (A) Western blot of HeLa cells transfected with control siRNA (siLuc) or siRNA specific for PRC1. The Western blot was probed with an anti-PRC1 antibody. α-Tubulin is shown as loading control. (B) IF for PLK1, PRC1, and α-tubulin of HeLa cells transfected with siLuc or siPRC1. DNA was visualized using DAPI. (C) DIC stills of a live cell imaging experiment of HeLa cells transfected with either siLuc or siPRC1 with (PLK1 inh.) or without addition of BI2536 (100 nM) before anaphase onset. Time point 00:00 (hours:minutes) refers to the first frame where we observed separating sisters. Stills of more time frames are shown in Fig. S1 B. (D) Percentage of cells showing either complete furrow ingression, full-furrow ingression followed by furrow regression, visible but minimal furrow ingression, or no furrow ingression (n = 100 cells imaged per condition). ****, P < 0.0001; χ² test for comparison of the indicated conditions; ns, not significant. One representative experiment out of two is shown. (E) Quantification of fluorescence intensity levels of RhoA at the equatorial cortex in anaphase as shown in F. Each dot represents an individual cell. Error bars depict the SD of the mean. ****, P < 0.0001; Student’s t test for comparison of the indicated conditions; ns, not significant. (F) IF for RhoA and Anillin of HeLa cells in anaphase transfected with the indicated siRNAs and treated with or without BI2536 (100 nM). Bars: 5 µm (B); 10 µm (C and F).