Figure S4.
BC inheritance during cytokinesis, functionality of the Rho biosensor in Can 10 cells, and expression, localization, and knockdown efficiency of Rho and ROCK isoforms in Can 10 cells, related to Figs. 4, 5, and 6. (A) Montage of time-lapse imaging of Can 10 cells expressing Mzt1-GFP and radixin–mScarlet during cytokinesis. Maximum-intensity projections are shown. The top and bottom panels show symmetric and asymmetric BC inheritance, respectively. White arrowheads at time 0 indicate spindle orientation parallel to the BC, resulting in BC inheritance into both daughters; yellow arrowheads at time 0 indicate spindle orientation perpendicular to the BC, resulting in BC inheritance into only one daughter. Note that Mzt1 brightens during cell division and relocates to the subapical region after cytokinesis. (B) Comparison of spindle orientation between symmetric and asymmetric BC inheritance. The combined scatter and box-and-whisker plots on the left show all cells expressing Mzt1-GFP and radixin–mScarlet; the plots on the right show spindle angle distributions relative to the BC for cells with symmetric or asymmetric BC inheritance (n = 46 cells for symmetric inheritance; n = 24 cells for asymmetric inheritance). Values are from two independent experiments and were analyzed using the Wilcoxon rank-sum test. (C) Expression of an active RhoA biosensor (dT-2×rGBD) does not impair polarization or BC formation in Can 10 cells. Top: schematic of the dT-2×rGBD construct. Bottom: quantification of Can 10 cells expressing the biosensor, categorized by polarity type and BC morphology. Data represent means ± SD from four independent experiments (≥268 cells per condition). (D) Quantification of RhoC expression. Data represent means ± SD from three independent experiments; ratios of RhoC/α-tubulin in control siRNA-transfected cells from each experiment are set to 1.0. (E) Immunoblot analysis of ROCK1- or ROCK2-depleted Can 10 cells. MWs of marker proteins are indicated in kDa. (F) Representative confocal images of Can 10 cells, cultured for 72 h, and stained with DAPI, phalloidin, and antibodies against ROCK2 and aPKC. Yellow arrows in the left panel indicate ROCK2 accumulation near the apical edge. Yellow arrowheads and double arrows in the right panels indicate ROCK2 accumulation at the edge of the BC and the midbody region, respectively. Scale bars, 5 µm (zoomed images in F), 10 µm (A and F). P value is indicated at the top of each graph; n.s., not significant. MWs, molecular weights. Source data are available for this figure: SourceData FS4. Refer to the image caption for details. Panel A shows time-lapse fluorescence images of Can 10 cells expressing Mzt1-GFP and Radixin-mScarlet during symmetric and asymmetric bile canaliculi inheritance. Panel B shows scatter plots and box plots comparing spindle angle distributions during symmetric and asymmetric bile canaliculi inheritance. Panel C shows a schematic of the dimeric Tomato–2xrGBD RhoA biosensor construct and bar graphs quantifying polarity and bile canaliculi formation. Panel D shows a bar graph quantifying normalized RhoC protein levels after siRNA treatments. Panel E shows immunoblot images analyzing ROCK1, ROCK2, RhoA, and α-tubulin expression after ROCK isoform knockdown. Panel F shows confocal fluorescence micrographs of Can 10 cells stained for ROCK2, aPKC, F-actin, and nuclei, highlighting ROCK2 localization near bile canaliculi and midbody regions.

BC inheritance during cytokinesis, functionality of the Rho biosensor in Can 10 cells, and expression, localization, and knockdown efficiency of Rho and ROCK isoforms in Can 10 cells, related to Figs. 4, 5, and 6. (A) Montage of time-lapse imaging of Can 10 cells expressing Mzt1-GFP and radixin–mScarlet during cytokinesis. Maximum-intensity projections are shown. The top and bottom panels show symmetric and asymmetric BC inheritance, respectively. White arrowheads at time 0 indicate spindle orientation parallel to the BC, resulting in BC inheritance into both daughters; yellow arrowheads at time 0 indicate spindle orientation perpendicular to the BC, resulting in BC inheritance into only one daughter. Note that Mzt1 brightens during cell division and relocates to the subapical region after cytokinesis. (B) Comparison of spindle orientation between symmetric and asymmetric BC inheritance. The combined scatter and box-and-whisker plots on the left show all cells expressing Mzt1-GFP and radixin–mScarlet; the plots on the right show spindle angle distributions relative to the BC for cells with symmetric or asymmetric BC inheritance (n = 46 cells for symmetric inheritance; n = 24 cells for asymmetric inheritance). Values are from two independent experiments and were analyzed using the Wilcoxon rank-sum test. (C) Expression of an active RhoA biosensor (dT-2×rGBD) does not impair polarization or BC formation in Can 10 cells. Top: schematic of the dT-2×rGBD construct. Bottom: quantification of Can 10 cells expressing the biosensor, categorized by polarity type and BC morphology. Data represent means ± SD from four independent experiments (≥268 cells per condition). (D) Quantification of RhoC expression. Data represent means ± SD from three independent experiments; ratios of RhoC/α-tubulin in control siRNA-transfected cells from each experiment are set to 1.0. (E) Immunoblot analysis of ROCK1- or ROCK2-depleted Can 10 cells. MWs of marker proteins are indicated in kDa. (F) Representative confocal images of Can 10 cells, cultured for 72 h, and stained with DAPI, phalloidin, and antibodies against ROCK2 and aPKC. Yellow arrows in the left panel indicate ROCK2 accumulation near the apical edge. Yellow arrowheads and double arrows in the right panels indicate ROCK2 accumulation at the edge of the BC and the midbody region, respectively. Scale bars, 5 µm (zoomed images in F), 10 µm (A and F). P value is indicated at the top of each graph; n.s., not significant. MWs, molecular weights. Source data are available for this figure: SourceData FS4.

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