Figure 8.
p190B maintains hepatic polarity by downregulating RhoA activity. (A) Venn diagram of ARHGAP proteins identified by BioID analysis. Their preferences for Rho family small GTPases are shown on the right. The RhoGAP specific to N-cadherin is indicated in red. Note that the E-cadherin–specific group does not include any ARHGAPs. n.d., not detected. (B) Localization of p190B in Can 10 cells. Representative confocal images of Can 10 cells expressing mScarlet–p190B are shown. Cells were cultured for 3 days, fixed, and stained with DAPI, phalloidin, and an anti-N-cadherin antibody. Yellow arrows indicate accumulation of p190B at N-cadherin–positive AJs. (C) Depletion of p190A and/or p190B does not alter RhoA levels. Immunoblot analysis of Can 10 cells transfected with p190A and/or p190B siRNAs. MWs of marker proteins are indicated in kDa. (D) p190B, but not p190A, is required for hepatic polarity maintenance. Shown are representative confocal images of Can 10 cells transfected with p190A and/or p190B siRNAs, cultured for 72 h, and stained with DAPI and antibodies against ZO-1 and aPKC. Yellow arrows indicate the apical membrane in columnar polarity cells. (E) Quantification of polarity and BC structures in cells from D. Data represent means ± SD from four independent experiments (≥431 cells per condition). (F and G) Elevated RhoA activity induced by p190B depletion. (F) Representative confocal images of Can 10 cells transfected with p190B siRNA and expressing dT-2×rGBD. Arrows indicate high RhoA activity at the apical edge in columnar polarity cells. (G) Product of the apical edge surface area with high probe activity and the mean intensity ratio of dT-2×rGBD at BCs versus cytoplasm in control or p190B-knockdown cells. Values are from two independent experiments (≥34 cells per condition). Values between the two groups were compared using the Wilcoxon rank-sum test. Scale bars, 10 µm (B and F), 20 µm (D). P values are indicated at the top of each graph; n.s., not significant. MWs, molecular weights. Source data are available for this figure: SourceData F8. Refer to the image caption for details. Panel A shows a Venn diagram comparing ARHGAP proteins associated with E-cadherin and N-cadherin complexes alongside preferred Rho family GTPase targets. Panel B shows confocal fluorescence micrographs of cells expressing mScarlet–p190B stained for N-cadherin, F-actin, and nuclei. Panel C shows immunoblot images analyzing p190A, p190B, RhoA, and α-tubulin expression after siRNA-mediated depletion. Panel D shows confocal fluorescence micrographs and orthogonal views of cells stained for ZO-1, aPKC, F-actin, and nuclei after p190A or p190B knockdown. Panel E shows bar graphs quantifying hepatic polarity, columnar polarity, and bile canaliculi structures after p190A or p190B depletion. Panel F shows confocal fluorescence micrographs of active RhoA biosensor localization in control and p190B-knockdown cells. Panel G shows box and whisker plots comparing apical RhoA activity and active RhoA intensity ratios between control and p190B-depleted cells.

p190B maintains hepatic polarity by downregulating RhoA activity. (A) Venn diagram of ARHGAP proteins identified by BioID analysis. Their preferences for Rho family small GTPases are shown on the right. The RhoGAP specific to N-cadherin is indicated in red. Note that the E-cadherin–specific group does not include any ARHGAPs. n.d., not detected. (B) Localization of p190B in Can 10 cells. Representative confocal images of Can 10 cells expressing mScarlet–p190B are shown. Cells were cultured for 3 days, fixed, and stained with DAPI, phalloidin, and an anti-N-cadherin antibody. Yellow arrows indicate accumulation of p190B at N-cadherin–positive AJs. (C) Depletion of p190A and/or p190B does not alter RhoA levels. Immunoblot analysis of Can 10 cells transfected with p190A and/or p190B siRNAs. MWs of marker proteins are indicated in kDa. (D) p190B, but not p190A, is required for hepatic polarity maintenance. Shown are representative confocal images of Can 10 cells transfected with p190A and/or p190B siRNAs, cultured for 72 h, and stained with DAPI and antibodies against ZO-1 and aPKC. Yellow arrows indicate the apical membrane in columnar polarity cells. (E) Quantification of polarity and BC structures in cells from D. Data represent means ± SD from four independent experiments (≥431 cells per condition). (F and G) Elevated RhoA activity induced by p190B depletion. (F) Representative confocal images of Can 10 cells transfected with p190B siRNA and expressing dT-2×rGBD. Arrows indicate high RhoA activity at the apical edge in columnar polarity cells. (G) Product of the apical edge surface area with high probe activity and the mean intensity ratio of dT-2×rGBD at BCs versus cytoplasm in control or p190B-knockdown cells. Values are from two independent experiments (≥34 cells per condition). Values between the two groups were compared using the Wilcoxon rank-sum test. Scale bars, 10 µm (B and F), 20 µm (D). P values are indicated at the top of each graph; n.s., not significant. MWs, molecular weights. Source data are available for this figure: SourceData F8.

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