FIGURE 3:

Grhl2 increases epithelial barrier function of HPPL by up-regulating Cldn3 and 4. (A) Adult mature cholangiocytes show stronger barrier functions than HPPL. Adult cholangiocytes were isolated as EpCAM⁺ cells and plated onto a culture insert coated with type I collagen gel. HPPL were plated on a culture insert coated with laminin. FITC-dextran, 4 kDa, 1 mg/ml was added inside the culture insert, and 2 h later the medium in the bottom was used for measuring the fluorescence of FITC. (B) Grhl2 decreases the efflux of 4-kDa FITC-dextran through the monolayer of HPPL. (C) PCR analysis for expression of Cldn2, 3, 4, 6, 7, and 8. Cldn3, 4, and 8 are transcriptionally up-regulated by overexpression of Grhl2. (D) Western blot analysis for expression of Cldn2, 3, 4, 6, 7, and 8. Grhl2 increases Cldn3 and 4 proteins as compared with the control. (E) Detection of Cldn3 and 4 in Triton X-100–soluble and –insoluble fractions. Grhl2 increases Cldn3 and 4 proteins both in Triton X-100–insoluble fraction, which represents Cldns incorporated into TJs, and in soluble fraction. (F, G) Localization of Cldn3 and 4 in 2D culture. Cldn3 and 4 (red) are colocalized with ZO1 (green), suggesting that they are incorporated into TJs in HPPL-Grhl2. The monolayer of HPPL was stained with anti-ZO1 and either anti-Cldn3 or 4 antibody.