Figure 1

Par3 expression affects YAP subcellular translocation in MDCK cells. (a) Distribution and co-localization of YAP (red), Par3 (green), a merged image with only YAP and Par3 and a merged image with 4′, 6-diamidino-2-phenylindole (blue) of MDCK II cells at different cell densities. Scale bar: 25 μm. (b) Ratios of the percentages of nuclear and cytoplasmic YAP and Par3 at different cell densities are shown. Pictures were analyzed with a Columbus Image Data Storage and Analysis System. (c) Representative images of YAP translocation into the cytoplasm at low cell density when Par3 was knocked down but not at high cell density. MDCK II cells were transfected with siRNA for Par3 at different cell densities. YAP (red), Par3 (green) and a merged image. Scale bar: 25 μm. All pictures were taken with a Leica TCS SP5 microscope. (d) The areas representing the cytosolic or nuclear fraction are indicated with bars at the top of each histogram. N: nucleus; C: cytoplasm. The histogram analysis of YAP and Par3 signal intensity in subcellular distributions was performed with the ‘Plot profile’ in ImageJ software. The analyzed area is indicated by the white straight line in the overview. YAP signal intensity distribution (red line), nucleus signal intensity distribution (blue line). (e, f) YAP translocated into the nucleus with Par3 overexpression, and YAP nuclear localization was inhibited when Par3 was knocked down. A cell fraction assay was performed after 293T cells were transfected with Flag-Par3 or MDCK II cells were transfected with siRNA for Par3 for 2 days at low cell density. Lamin-B is the nuclear marker and β-actin is the cytoplasmic marker. (g) Par3 knockdown reduced YAP translocation to the nucleus in the Ca²⁺ off switch system. Representative images of YAP translocation into the nucleus at high cell density when Ca²⁺ was depleted are shown. After 2 days, while MDCK II cells were transfected with siRNA for Par3, MDCK II cells were cultured with Ca²⁺-free medium for the indicated time course. YAP (red), Par3 (green) and ZO-1 (purple). Scale bar: 25 μm. (h) Ratios of the percentages of nuclear and cytoplasmic YAP and Par3 at different time points. Pictures were analyzed with a Columbus Image Data Storage and Analysis System. **P<0.01, *P<0.05.