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. 2018 Mar 14;14(3):e1006916. doi: 10.1371/journal.ppat.1006916

Fig 3. TACSTD2 gene silencing disrupts CLDN1 and OCLN cellular localization in hepatoma cells and primary human hepatocytes.

Fig 3

(A) Visualization of TACSTD2 (green) and CLDN1 (red) in parental Huh7.5 cells transfected with siTACSTD2 or an irrelevant siRNA (siControl). CLDN1 appears speckled and fragmented in siTACSTD2-treated cells but maintains its regular linear pattern along the cellular membrane in siControl-treated cells. (B) Visualization of TACSTD2 (green) and OCLN (red) in parental Huh7.5 cells transfected with siTACSTD2 or siControl. OCLN appears speckled and fragmented in siTACSTD2-treated cells but maintains its regular linear pattern along the cellular membrane in siControl-treated cells. (C) Visualization of CLDN1 and OCLN in primary human hepatocytes transfected with siTACSTD2 or siControl. Both CLDN1 and OCLN appear speckled and fragmented in siTACSTD2-treated cells but maintain their regular linear distribution along the cellular membrane in siControl-treated cells. (D) TACSTD2 gene silencing results in the reduction of phosphorylation levels of CLDN1 and OCLN in both parental and TACSTD2-overexpressing Huh7.5 cells. Cell lysates from both parental and TACSTD2-overexpressing Huh7.5 cells were immunoprecipitated with anti-CLDN1 and anti-OCLN antibodies or control IgG, at 72h after transfection with either siControl or siTACSTD2. Phosphorylated CLDN1 and OCLN were detected using an anti-PKC substrate-specific antibody; total CLDN1 and total OCLN were detected using anti-CLDN1 and anti-OCLN specific antibodies, respectively. Whole cellular lysates were simultaneously subjected to immunoblotting using anti-CLDN1 and anti-OCLN antibodies. (E) RT-PCR quantification of TACSTD2 in siControl- and siTACSTD2-transfected cells, expressed as 2-ΔΔCT, where ΔΔCT is the average difference between the siTACSTD2 ΔCT and siControl ΔCT.