Figure 8.
Co-depletion of NuMA and Prc1 affect cytokinetic furrow initiation. (A–D) Confocal live-imaging analysis in combination with differential interphase contrast (DIC) microscopy images of HeLa Kyoto cells stably expressing mCherry-H2B, and were either transfected with control siRNA (A; Video 1), siRNA against NuMA (B; Video 2), siRNA against Prc1 (C; Video 3), or siRNA against Prc1 and NuMA (D(i); Video 4 and D(ii); Video 5). D(i) represents cells where the cytokinetic furrow was initiated but did not fully ingress and regress back before complete ingression (∼20% of the cells). In contrast, D(ii) represents cells where no cytokinetic furrow initiation was observed (∼10% of the cells). The recording was started 50 h post-transfection. “0 min” time-point represents metaphase to anaphase transition. The white arrowhead represents appearance of cytokinetic furrow. The yellow asterisk depicts cytokinesis failure (binucleated cell) in Prc1 siRNA transfected cell after complete furrow ingression, followed by regression. The scale bar in these panels represents 10 μm. (E) Quantification of various cytokinetic phenotypes as indicated in HeLa Kyoto cells stably expressing mCherry-H2B that were transfected with Prc1 siRNA or Prc1 and NuMA siRNA (n > 50 cells from three independent experiments). The recording was started 50 h post-transfection. “0 min” time-point represents metaphase to anaphase transition, and for every cell time-lapse, movies were recorded till 70 min from time 0 min. Since control siRNA transfected cells, and NuMA siRNA transfected cells do not reveal any furrow regression and cytokinesis failure within 70 min of time-lapse movies acquisition, their data are not included in the graph. (Total cells analyzed for Prc1 siRNA and for NuMA and Prc1 siRNA transfected cells were 51 and 87, respectively). (F–J) Schematic representation of anaphase cell depicting the method of measurements of the cytokinetic furrow width (d [µm]) from time-lapse confocal live imaging obtained for A–D and F. Furrow width measurements in cells stably expressing mCherry-H2B that were transfected with control siRNA (G), siRNA against NuMA (H), siRNA against Prc1 (I), and siRNA against Prc1 and NuMA (J). Please note that in contrast to Prc1 siRNA transfected cells, 10% of cells transfected with NuMA and Prc1 siRNA do not initiate cytokinetic furrow ingression, while 20% of these cells do not fully ingress cytokinetic furrow and proceed with furrow regression in time <35 min from time “0 min” that indicates metaphase to anaphase transition. (a minimum of 16 cells were used for such analysis for each siRNA condition, as depicted on the figure panels).

Co-depletion of NuMA and Prc1 affect cytokinetic furrow initiation. (A–D) Confocal live-imaging analysis in combination with differential interphase contrast (DIC) microscopy images of HeLa Kyoto cells stably expressing mCherry-H2B, and were either transfected with control siRNA (A; Video 1), siRNA against NuMA (B; Video 2), siRNA against Prc1 (C; Video 3), or siRNA against Prc1 and NuMA (D(i); Video 4 and D(ii); Video 5). D(i) represents cells where the cytokinetic furrow was initiated but did not fully ingress and regress back before complete ingression (∼20% of the cells). In contrast, D(ii) represents cells where no cytokinetic furrow initiation was observed (∼10% of the cells). The recording was started 50 h post-transfection. “0 min” time-point represents metaphase to anaphase transition. The white arrowhead represents appearance of cytokinetic furrow. The yellow asterisk depicts cytokinesis failure (binucleated cell) in Prc1 siRNA transfected cell after complete furrow ingression, followed by regression. The scale bar in these panels represents 10 μm. (E) Quantification of various cytokinetic phenotypes as indicated in HeLa Kyoto cells stably expressing mCherry-H2B that were transfected with Prc1 siRNA or Prc1 and NuMA siRNA (n > 50 cells from three independent experiments). The recording was started 50 h post-transfection. “0 min” time-point represents metaphase to anaphase transition, and for every cell time-lapse, movies were recorded till 70 min from time 0 min. Since control siRNA transfected cells, and NuMA siRNA transfected cells do not reveal any furrow regression and cytokinesis failure within 70 min of time-lapse movies acquisition, their data are not included in the graph. (Total cells analyzed for Prc1 siRNA and for NuMA and Prc1 siRNA transfected cells were 51 and 87, respectively). (F–J) Schematic representation of anaphase cell depicting the method of measurements of the cytokinetic furrow width (d [µm]) from time-lapse confocal live imaging obtained for A–D and F. Furrow width measurements in cells stably expressing mCherry-H2B that were transfected with control siRNA (G), siRNA against NuMA (H), siRNA against Prc1 (I), and siRNA against Prc1 and NuMA (J). Please note that in contrast to Prc1 siRNA transfected cells, 10% of cells transfected with NuMA and Prc1 siRNA do not initiate cytokinetic furrow ingression, while 20% of these cells do not fully ingress cytokinetic furrow and proceed with furrow regression in time <35 min from time “0 min” that indicates metaphase to anaphase transition. (a minimum of 16 cells were used for such analysis for each siRNA condition, as depicted on the figure panels).

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