Figure 9.
ARVCF mediates the role of N-cadherin in hepatic polarity maintenance by downregulating RhoA activity. (A) Localization of p120-catenin and ARVCF during interphase and cytokinesis. Shown are representative iSIM images of Can 10 cells cultured for 3 days and stained with DAPI, phalloidin, and antibodies against p120-catenin and ARVCF. Yellow arrowheads and arrows indicate AJs and the cleavage furrow, respectively. (B) N-cadherin, but not E-cadherin, preferentially associates with ARVCF over p120-catenin. Lysates of Can 10 cells treated with the reversible cross-linker DSP were immunoprecipitated using control IgG, anti-E-cadherin, or anti-N-cadherin antibody, followed by SDS-PAGE and immunoblot analysis. The asterisk indicates nonspecific bands in the lysates detected by the anti-ARVCF antibody. MWs of marker proteins are indicated in kDa. (C) Quantification of ARVCF and p120-catenin binding preference for E- and N-cadherin. Top: intensities of pulled-down cadherins (left) and their normalized protein amounts (right). Bottom: ratios of coprecipitated ARVCF (left) or ARVCF/p120-catenin (right) normalized to the cadherin pulldown. Data represent means ± SD from three independent experiments. The ARVCF/N-cadherin ratio (left) and the ARVCF/p120-catenin ratio in N-cadherin pulldowns (right) are set to 1.0. See also Fig. S1 H. (D) Knockdown of p120, but not ARVCF, destabilizes E-cadherin without affecting N-cadherin. Left: representative immunoblots of single and double knockdown cells. The asterisk indicates nonspecific bands in the lysates detected by the anti-ARVCF antibody. Right: quantification of E-cadherin-to-N-cadherin ratios. Data represent means ± SD from three independent experiments; the ratio in control siRNA-transfected cells is set to 1.0. (E) ARVCF primarily regulates hepatic polarity, while p120-catenin affects BC elongation. Shown are representative confocal images of Can 10 cells transfected with ARVCF and/or p120-catenin siRNAs, cultured for 72 h, and stained with DAPI and antibodies against p120-catenin and aPKC. Yellow arrows indicate the apical membrane in columnar polarity cells. (F) Quantification of polarity and BC structures in cells from E. Data represent means ± SD from four independent experiments (≥803 cells per condition). (G and H) N-cadherin depletion increases ROCK2 accumulation at the apical edge. (G) Representative confocal images of Can 10 cells transfected with control or N-cadherin siRNAs, cultured for 72 h, and stained with DAPI and antibodies against aPKC and ROCK2. Arrows indicate ROCK2 accumulation on the apical-most junctions. (H) Quantification of ROCK2 intensity at the apical edge vs. cytoplasm. Data are from two independent experiments (≥54 cells per condition). (I and J) ROCK2 accumulates at AJs in ARVCF/p120-catenin double knockdown cells. Shown are representative confocal images of Can 10 cells transfected with both siRNAs, cultured for 72 h, and stained with DAPI and antibodies against aPKC and ROCK2. Arrows indicate ROCK2 accumulation on the apical-most junctions. (J) Quantification of ROCK2 localization at AJs. Ratios of ROCK2 intensity at the apical edge vs. cytoplasm are shown for control and double knockdown cells. Data are from two independent experiments (≥51 cells per condition). (K and L) Elevated Rho activity induced by double depletion of ARVCF/p120-catenin. (K) Representative confocal images of Can 10 cells transfected with both siRNAs and expressing dT-2×rGBD. Arrows indicate high RhoA activity at the apical edge in columnar polarity cells. (L) 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 the double knockdown cells. Values are from two independent experiments (≥32 cells per condition). Values between the two groups were compared using the Wilcoxon rank-sum test. Scale bars, 5 µm (A), 10 µm (K), 20 µm (E, G, and I). 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 F9. Refer to the image caption for details. Panel A shows super-resolution fluorescence micrographs of p120-catenin and ARVCF localization during interphase and cytokinesis in cultured cells. Panel B shows immunoblot images from co-immunoprecipitation assays analyzing ARVCF and p120-catenin associations with E-cadherin and N-cadherin. Panel C shows bar graphs quantifying cadherin pulldown efficiency and ARVCF association relative to p120-catenin binding. Panel D shows immunoblot images and bar graphs analyzing E-cadherin and N-cadherin expression after p120-catenin and ARVCF knockdown. Panel E shows confocal fluorescence micrographs and orthogonal views of cells stained for p120-catenin, aPKC, and nuclei after single or double knockdown treatments. Panel F shows bar graphs quantifying hepatic polarity, columnar polarity, and bile canaliculi structures after p120-catenin and ARVCF depletion. Panel G shows confocal fluorescence micrographs and orthogonal views of ROCK2 localization in control and N-cadherin–depleted cells stained for aPKC and nuclei. Panel H shows box-and-whisker plots quantifying ROCK2 intensity at apical edges relative to cytoplasm after N-cadherin depletion. Panel I shows confocal fluorescence micrographs and orthogonal views of ROCK2 localization after combined p120-catenin and ARVCF knockdown. Panel J shows box-and-whisker plots quantifying ROCK2 accumulation at apical junctions relative to cytoplasm after double knockdown treatment. Panel K shows confocal fluorescence micrographs of active RhoA biosensor localization in control and double knockdown cells. Panel L shows box-and-whisker plots quantifying apical edge surface area with elevated active RhoA biosensor intensity after double knockdown treatment.

ARVCF mediates the role of N-cadherin in hepatic polarity maintenance by downregulating RhoA activity. (A) Localization of p120-catenin and ARVCF during interphase and cytokinesis. Shown are representative iSIM images of Can 10 cells cultured for 3 days and stained with DAPI, phalloidin, and antibodies against p120-catenin and ARVCF. Yellow arrowheads and arrows indicate AJs and the cleavage furrow, respectively. (B) N-cadherin, but not E-cadherin, preferentially associates with ARVCF over p120-catenin. Lysates of Can 10 cells treated with the reversible cross-linker DSP were immunoprecipitated using control IgG, anti-E-cadherin, or anti-N-cadherin antibody, followed by SDS-PAGE and immunoblot analysis. The asterisk indicates nonspecific bands in the lysates detected by the anti-ARVCF antibody. MWs of marker proteins are indicated in kDa. (C) Quantification of ARVCF and p120-catenin binding preference for E- and N-cadherin. Top: intensities of pulled-down cadherins (left) and their normalized protein amounts (right). Bottom: ratios of coprecipitated ARVCF (left) or ARVCF/p120-catenin (right) normalized to the cadherin pulldown. Data represent means ± SD from three independent experiments. The ARVCF/N-cadherin ratio (left) and the ARVCF/p120-catenin ratio in N-cadherin pulldowns (right) are set to 1.0. See also Fig. S1 H. (D) Knockdown of p120, but not ARVCF, destabilizes E-cadherin without affecting N-cadherin. Left: representative immunoblots of single and double knockdown cells. The asterisk indicates nonspecific bands in the lysates detected by the anti-ARVCF antibody. Right: quantification of E-cadherin-to-N-cadherin ratios. Data represent means ± SD from three independent experiments; the ratio in control siRNA-transfected cells is set to 1.0. (E) ARVCF primarily regulates hepatic polarity, while p120-catenin affects BC elongation. Shown are representative confocal images of Can 10 cells transfected with ARVCF and/or p120-catenin siRNAs, cultured for 72 h, and stained with DAPI and antibodies against p120-catenin and aPKC. Yellow arrows indicate the apical membrane in columnar polarity cells. (F) Quantification of polarity and BC structures in cells from E. Data represent means ± SD from four independent experiments (≥803 cells per condition). (G and H) N-cadherin depletion increases ROCK2 accumulation at the apical edge. (G) Representative confocal images of Can 10 cells transfected with control or N-cadherin siRNAs, cultured for 72 h, and stained with DAPI and antibodies against aPKC and ROCK2. Arrows indicate ROCK2 accumulation on the apical-most junctions. (H) Quantification of ROCK2 intensity at the apical edge vs. cytoplasm. Data are from two independent experiments (≥54 cells per condition). (I and J) ROCK2 accumulates at AJs in ARVCF/p120-catenin double knockdown cells. Shown are representative confocal images of Can 10 cells transfected with both siRNAs, cultured for 72 h, and stained with DAPI and antibodies against aPKC and ROCK2. Arrows indicate ROCK2 accumulation on the apical-most junctions. (J) Quantification of ROCK2 localization at AJs. Ratios of ROCK2 intensity at the apical edge vs. cytoplasm are shown for control and double knockdown cells. Data are from two independent experiments (≥51 cells per condition). (K and L) Elevated Rho activity induced by double depletion of ARVCF/p120-catenin. (K) Representative confocal images of Can 10 cells transfected with both siRNAs and expressing dT-2×rGBD. Arrows indicate high RhoA activity at the apical edge in columnar polarity cells. (L) 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 the double knockdown cells. Values are from two independent experiments (≥32 cells per condition). Values between the two groups were compared using the Wilcoxon rank-sum test. Scale bars, 5 µm (A), 10 µm (K), 20 µm (E, G, and I). 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 F9.

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