Figure 2.
Increased E-cadherin expression relative to N-cadherin induces hepatic polarity. (A) Representative iSIM images of polarized Can 10 cells and unpolarized parental Fao cells. Cells were cultured for 3 days, then fixed, and stained with DAPI and antibodies against MDR1 and ZO-1. Yellow arrowheads indicate nonpolarized cells lacking MDR1- and ZO-1–positive structures. (B) Immunoblot analysis of E- and N-cadherin expression in Fao and Can 10 cells. Immunoblotting was performed using antibodies against N-cadherin, E-cadherin, p120-catenin, and α-tubulin. MWs of marker proteins are indicated in kDa. (C) Normalized intensity ratios of N-cadherin, E-cadherin, and p120-catenin relative to tubulin in Fao and Can 10 cells. Values represent means ± SD from three independent experiments shown in B. Ratios in Fao cells were set to 1.0. See also Fig. S1, G and H. (D) Analysis of cell polarization in Can 10 and Fao cells. Cells were cultured for 3 days, then fixed, and stained with phalloidin, DAPI, and antibodies against radixin and ZO-1. iSIM images were shown in horizontal (xy) and orthogonal (xz) views. Note that the Fao cells indicated by yellow arrowheads contain phalloidin-positive structures located within the cells. (E) Quantification of Can 10 and Fao cells with various types of polarity and BC structures. Values are means ± SD from four independent experiments (≥401 cells counted per sample). (F) Analysis of TJ protein localization and assembly in Can 10 and Fao cells. Cells were cultured for 3 days, then fixed, and stained with DAPI and antibodies against Par-3 and ZO-1. Representative iSIM images are shown. Yellow arrows indicate small TJ-like structures between 2 cells. (G) Analysis of AJ protein localization and assembly in relation to F-actin in Can 10 and Fao cells. Cells were cultured for 3 days, then fixed, and stained with phalloidin, DAPI, and antibodies against E-cadherin and N-cadherin. Representative iSIM images are shown. (H) Induction of hepatic polarity in Fao cells by increasing E-cadherin expression. Fao cells were cultured for 4 days following transduction with lentiviruses expressing either mScarlet or E-cadherin–mScarlet, then fixed, and stained with antibodies against radixin and ZO-1. Representative confocal images of unpolarized cells expressing mScarlet only, or polarized cells with pBC or tBC expressing E-cadherin–mScarlet, are shown. (I) Quantification of Fao cells expressing mScarlet alone or E-cadherin–mScarlet with indicated polarity and BC structures. Values represent means ± SD from four independent experiments (≥154 cells counted per sample). (J) Quantification of ZO-1 length in Fao cells expressing mScarlet only or E-cadherin–mScarlet. Values are from four independent experiments (≥211 ZO-1–positive structures counted per condition). Scale bars, 10 µm (A, D, F, G, and H). P values are indicated at the top of each graph. n.s., not significant. TJ, tight junction; pBC, primordial BC; tBC, tubular BC; MWs, molecular weights. Source data are available for this figure: SourceData F2. Refer to the image caption for details. Panel A shows super-resolution fluorescence micrographs comparing polarized Can 10 cells and unpolarized Fao cells stained for MDR1, ZO-1, and nuclei. Panel B shows immunoblot images analyzing E-cadherin, N-cadherin, p120-catenin, and alpha-tubulin expression in Fao and Can 10 cells. Panel C shows bar graphs comparing normalized cadherin and p120-catenin protein expression levels between Fao and Can 10 cells. Panel D shows fluorescence micrographs and orthogonal views of Can 10 and Fao cells stained for ZO-1, radixin, F-actin, and nuclei to assess cell polarity. Panel E shows bar graphs quantifying hepatic polarity, columnar polarity, and bile canaliculi types in Fao and Can 10 cells. Panel F shows fluorescence micrographs of Par-3 and ZO-1 localization in Can 10 and Fao cells. Panel G shows fluorescence micrographs of E-cadherin, N-cadherin, F-actin, and nuclei illustrating adherens junction organization in cultured cells. Panel H shows confocal micrographs of Fao cells expressing mScarlet or E-cadherin–mScarlet stained for radixin and ZO-1. Panel I shows bar graphs quantifying hepatic polarity and bile canaliculi formation in cells expressing mScarlet or E-cadherin–mScarlet. Panel J shows a scatter plot comparing ZO-1 structure lengths between control and E-cadherin–mScarlet expressing cells.

Increased E-cadherin expression relative to N-cadherin induces hepatic polarity. (A) Representative iSIM images of polarized Can 10 cells and unpolarized parental Fao cells. Cells were cultured for 3 days, then fixed, and stained with DAPI and antibodies against MDR1 and ZO-1. Yellow arrowheads indicate nonpolarized cells lacking MDR1- and ZO-1–positive structures. (B) Immunoblot analysis of E- and N-cadherin expression in Fao and Can 10 cells. Immunoblotting was performed using antibodies against N-cadherin, E-cadherin, p120-catenin, and α-tubulin. MWs of marker proteins are indicated in kDa. (C) Normalized intensity ratios of N-cadherin, E-cadherin, and p120-catenin relative to tubulin in Fao and Can 10 cells. Values represent means ± SD from three independent experiments shown in B. Ratios in Fao cells were set to 1.0. See also Fig. S1, G and H. (D) Analysis of cell polarization in Can 10 and Fao cells. Cells were cultured for 3 days, then fixed, and stained with phalloidin, DAPI, and antibodies against radixin and ZO-1. iSIM images were shown in horizontal (xy) and orthogonal (xz) views. Note that the Fao cells indicated by yellow arrowheads contain phalloidin-positive structures located within the cells. (E) Quantification of Can 10 and Fao cells with various types of polarity and BC structures. Values are means ± SD from four independent experiments (≥401 cells counted per sample). (F) Analysis of TJ protein localization and assembly in Can 10 and Fao cells. Cells were cultured for 3 days, then fixed, and stained with DAPI and antibodies against Par-3 and ZO-1. Representative iSIM images are shown. Yellow arrows indicate small TJ-like structures between 2 cells. (G) Analysis of AJ protein localization and assembly in relation to F-actin in Can 10 and Fao cells. Cells were cultured for 3 days, then fixed, and stained with phalloidin, DAPI, and antibodies against E-cadherin and N-cadherin. Representative iSIM images are shown. (H) Induction of hepatic polarity in Fao cells by increasing E-cadherin expression. Fao cells were cultured for 4 days following transduction with lentiviruses expressing either mScarlet or E-cadherin–mScarlet, then fixed, and stained with antibodies against radixin and ZO-1. Representative confocal images of unpolarized cells expressing mScarlet only, or polarized cells with pBC or tBC expressing E-cadherin–mScarlet, are shown. (I) Quantification of Fao cells expressing mScarlet alone or E-cadherin–mScarlet with indicated polarity and BC structures. Values represent means ± SD from four independent experiments (≥154 cells counted per sample). (J) Quantification of ZO-1 length in Fao cells expressing mScarlet only or E-cadherin–mScarlet. Values are from four independent experiments (≥211 ZO-1–positive structures counted per condition). Scale bars, 10 µm (A, D, F, G, and H). P values are indicated at the top of each graph. n.s., not significant. TJ, tight junction; pBC, primordial BC; tBC, tubular BC; MWs, molecular weights. Source data are available for this figure: SourceData F2.

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