RhoA is the major isoform responsible for BC elongation. (A) RhoA accumulates at the BC region in polarized Can 10 cells but not in unpolarized parental Fao cells. Left: representative confocal images of Can 10 and Fao cells cultured for 3 days and stained with DAPI and antibodies against RhoA and ZO-1. Middle: quantification of RhoA intensity at ZO-1–positive PMs (left) or lateral membranes (right), normalized to cytoplasmic intensity. Values are from two independent experiments (≥50 cells per condition). The intensity ratios were analyzed by the Wilcoxon rank-sum test. Right: immunoblot analysis and quantification of RhoA levels in Fao and Can 10 cells. Data represent means ± SD from three independent experiments; RhoA/α-tubulin ratio in Fao cells is normalized to 1.0. Normalized RhoA levels were analyzed using Welch’s t test. (B and C) RhoA biosensor accumulates at the apical region and cleavage furrow during cytokinesis in cells with a preexisting BC. (B) Montage of time-lapse imaging from Can 10 cells expressing dT-2×rGBD during cytokinesis. SUM projection and confocal images are shown. Zoomed views of the boxed regions in the confocal images are also shown (bottom right). The arrow in the SUM image at time −50 indicates a preexisting BC; the arrowhead in the zoomed image at time 0 indicates the midbody. See also Video 1. (C) dT-2×rGBD intensities at the BC region over time from cells in (B) (n = 37) were quantified and plotted. Bold lines and shaded bands represent the mean and SD, respectively, from two independent experiments, as throughout this study. (D–F) RhoA is required for BC elongation. (D) Immunoblot analysis of RhoA knockdown in Can 10 cells. MWs of marker proteins are indicated in kDa. (E) Representative confocal images of RhoA-depleted Can 10 cells stained with DAPI and antibodies against RhoA and pERM. (F) Quantification of polarity and BC features in RhoA-knockdown cells. Data represent means ± SD from four independent experiments (≥593 cells per condition). (G) Expression of RhoA in hepatoblasts and hepatocytes during liver development. Liver sections from different developmental stages were immunostained with antibodies against RhoA and E-cadherin, along with DAPI staining. Yellow arrowheads show the colocalization of RhoA with E-cadherin. (H) ScRNA-seq analysis of Rho subfamily genes during mouse liver development. Violin plots represent the expression of RhoA, RhoB, and RhoC in hepatoblasts and hepatocytes from the scRNA-seq data (Yang et al., 2017). Each dot represents a single cell. The black line within each violin plot indicates the median expression level. (I) Knockdown of RhoA increases RhoB expression. Left: representative immunoblots of cells depleted of RhoA, RhoB, or RhoC. Right: quantification of RhoA and RhoB protein levels. Data represent means ± SD from three independent experiments; Rho/α-tubulin ratio in control siRNA-transfected cells from each experiment is set to 1.0. See also Fig. S4 D. (J) Localization of RhoB in RhoA-depleted cells. Shown are representative confocal images of RhoA-depleted Can 10 cells stained with DAPI, phalloidin, and antibodies against RhoB and aPKC. Note that RhoB localizes to the PM and midbody only in RhoA-knockdown cells. (K) RhoA is the primary isoform responsible for apical Rho activity in Can 10 cells. Left: representative confocal images of dT-2×rGBD–expressing Can 10 cells transfected with siRNAs targeting Rho subfamily members. Arrowheads indicate BCs. Right: ratios of dT-2×rGBD intensities at the BC vs. cytoplasm in control and Rho-knockdown cells. Data are from two independent experiments (≥32 cells per condition). Values across conditions were analyzed by the Wilcoxon rank-sum test with Holm’s correction. (L and M) RhoA is the primary isoform responsible for BC elongation. (L) Representative confocal images of Can 10 cells transfected with control siRNA or siRNAs targeting Rho subfamily members. Cells were stained with DAPI, phalloidin, and anti-aPKC antibody. (M) Quantification of polarity and BC features in Rho subfamily-knockdown cells. Data represent means ± SD from four independent experiments (≥218 cells per condition). Scale bars, 2 µm (zoomed images in B), 10 µm (A, B, G, J, and K), 20 µm (E and L). P values are indicated in each graph; n.s., not significant. MWs, molecular weights; pERM, phospho-ezrin/radixin/moesin. Source data are available for this figure: SourceData F5.