Figure 3.
Distinct roles of E- and N-cadherin in BC elongation and hepatic polarity maintenance. (A–C) E-cadherin is required for BC elongation. (A) Immunoblot analysis of Can 10 cell lysates transfected with siRNAs targeting E-cadherin. MWs of marker proteins are indicated in kDa. (B) Representative confocal images of Can 10 cells transfected with E-cadherin siRNAs, cultured for 72 h, and stained with DAPI and antibodies against radixin and E-cadherin. The orthogonal views are shown in xz. (C) Quantification of various polarity and BC structures in siRNA-transfected cells. Data represent means ± SD from four independent experiments (≥480 cells per sample). (D) Restoration of tubular BC structures by rescue expression of E-cadherin. Can 10 cells were transfected with E-cadherin siRNA #1 one day earlier and then transduced with lentiviruses expressing either GFP or E-cadherin–GFP. Cells were fixed and stained with antibodies against E-cadherin and ZO-1, along with phalloidin. Top: representative confocal images are shown. Bottom: quantification of hepatic polarity and tubular BC structure in the indicated cells. Data represent means ± SD from four independent experiments (≥96 cells per sample). (E–G) N-cadherin is required for maintaining hepatic polarity. Experimental procedures and image analyses were as described in A–C, except N-cadherin siRNAs were used. Yellow arrows indicate the apical membrane in columnar polarity cells. Data represent means ± SD from four independent experiments (≥535 cells per sample). (H) E- and N-cadherin preferentially form homodimers or oligomers in Can 10 cells. Top: lysates and anti-GFP immunoprecipitates from Can 10 cells expressing GFP-tagged E-cadherin or N-cadherin, treated with the reversible cross-linker DSP, were analyzed by immunoblotting using antibodies against E-cadherin, N-cadherin, or GFP. MWs of marker proteins are indicated in kDa. Middle and Bottom: quantification of coprecipitated endogenous cadherins normalized to pulled-down GFP-tagged E- and N-cadherin, respectively. Data represent means ± SD from three independent experiments. (I) Super-resolution imaging of E- and N-cadherin localization. STED microscopy of Can 10 cells cultured for 3 days, fixed, and stained with antibodies against E- and N-cadherin. Lower panels show magnified views of boxed regions. Yellow arrowheads indicate E-cadherin–positive vesicles. Yellow arrows indicate the E-cadherin–positive leading edge of a cell. (J) Dynamic localization of E- and N-cadherin during mitosis and cytokinesis. Can 10 cells cultured for 72 h were fixed and stained with phalloidin, DAPI, and antibodies against E- and N-cadherin. Representative confocal images are shown. Arrowheads, arrows, and double arrows indicate AJs, polar cortex, and the basal cleavage furrow or midbody, respectively. See also Fig. S2 D. (K) Quantitative analysis of cadherin distribution at the PM during mitosis and cytokinesis. E- and N-cadherin intensities at lateral (top) or basal (bottom) PM regions were normalized to intensity at AJs. Data are from two independent experiments (≥21 cells per condition). Scale bars, 1 µm (lower panels in I), 5 µm (J), 10 µm (upper panels in I), 20 µm (B, D, and F). P values are indicated in each graph; n.s., not significant. MWs, molecular weights. Source data are available for this figure: SourceData F3. Refer to the image caption for details. Panel A shows immunoblot images of Can 10 cell lysates after E-cadherin siRNA treatment, analyzing cadherin and p120-catenin expression. Panel B shows confocal fluorescence micrographs and orthogonal views of E-cadherin–depleted cells stained for radixin, E-cadherin, and nuclei. Panel C shows bar graphs quantifying hepatic polarity, columnar polarity, and bile canaliculi structures after E-cadherin knockdown. Panel D shows confocal fluorescence micrographs and bar graphs illustrating restoration of tubular bile canaliculi structures by E-cadherin–GFP rescue expression. Panel E shows immunoblot images of Can 10 cell lysates after N-cadherin siRNA treatment, analyzing cadherin and p120-catenin expression. Panel F shows confocal fluorescence micrographs and orthogonal views of N-cadherin–depleted cells stained for radixin, N-cadherin, and nuclei. Panel G shows bar graphs quantifying polarity states and bile canaliculi structures after N-cadherin knockdown. Panel H shows immunoblot images and bar graphs analyzing homodimer or oligomer formation between E-cadherin and N-cadherin proteins. Panel I shows STED super-resolution micrographs of E-cadherin and N-cadherin localization with enlarged views highlighting vesicles and leading edges. Panel J shows confocal fluorescence micrographs of E-cadherin and N-cadherin distribution during mitosis and cytokinesis in cultured cells. Panel K shows box plots quantifying cadherin intensity distributions at plasma membrane regions during mitosis and cytokinesis.

Distinct roles of E- and N-cadherin in BC elongation and hepatic polarity maintenance. (A–C) E-cadherin is required for BC elongation. (A) Immunoblot analysis of Can 10 cell lysates transfected with siRNAs targeting E-cadherin. MWs of marker proteins are indicated in kDa. (B) Representative confocal images of Can 10 cells transfected with E-cadherin siRNAs, cultured for 72 h, and stained with DAPI and antibodies against radixin and E-cadherin. The orthogonal views are shown in xz. (C) Quantification of various polarity and BC structures in siRNA-transfected cells. Data represent means ± SD from four independent experiments (≥480 cells per sample). (D) Restoration of tubular BC structures by rescue expression of E-cadherin. Can 10 cells were transfected with E-cadherin siRNA #1 one day earlier and then transduced with lentiviruses expressing either GFP or E-cadherin–GFP. Cells were fixed and stained with antibodies against E-cadherin and ZO-1, along with phalloidin. Top: representative confocal images are shown. Bottom: quantification of hepatic polarity and tubular BC structure in the indicated cells. Data represent means ± SD from four independent experiments (≥96 cells per sample). (E–G) N-cadherin is required for maintaining hepatic polarity. Experimental procedures and image analyses were as described in A–C, except N-cadherin siRNAs were used. Yellow arrows indicate the apical membrane in columnar polarity cells. Data represent means ± SD from four independent experiments (≥535 cells per sample). (H) E- and N-cadherin preferentially form homodimers or oligomers in Can 10 cells. Top: lysates and anti-GFP immunoprecipitates from Can 10 cells expressing GFP-tagged E-cadherin or N-cadherin, treated with the reversible cross-linker DSP, were analyzed by immunoblotting using antibodies against E-cadherin, N-cadherin, or GFP. MWs of marker proteins are indicated in kDa. Middle and Bottom: quantification of coprecipitated endogenous cadherins normalized to pulled-down GFP-tagged E- and N-cadherin, respectively. Data represent means ± SD from three independent experiments. (I) Super-resolution imaging of E- and N-cadherin localization. STED microscopy of Can 10 cells cultured for 3 days, fixed, and stained with antibodies against E- and N-cadherin. Lower panels show magnified views of boxed regions. Yellow arrowheads indicate E-cadherin–positive vesicles. Yellow arrows indicate the E-cadherin–positive leading edge of a cell. (J) Dynamic localization of E- and N-cadherin during mitosis and cytokinesis. Can 10 cells cultured for 72 h were fixed and stained with phalloidin, DAPI, and antibodies against E- and N-cadherin. Representative confocal images are shown. Arrowheads, arrows, and double arrows indicate AJs, polar cortex, and the basal cleavage furrow or midbody, respectively. See also Fig. S2 D. (K) Quantitative analysis of cadherin distribution at the PM during mitosis and cytokinesis. E- and N-cadherin intensities at lateral (top) or basal (bottom) PM regions were normalized to intensity at AJs. Data are from two independent experiments (≥21 cells per condition). Scale bars, 1 µm (lower panels in I), 5 µm (J), 10 µm (upper panels in I), 20 µm (B, D, and F). P values are indicated in each graph; n.s., not significant. MWs, molecular weights. Source data are available for this figure: SourceData F3.

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