Fig. 2

YAP restricts trophoblast stem cell proliferation. A BrdU incorporation assay in TSCs transfected with scramble or YAP siRNA. B Photomicrographic images of BrdU incorporated cells (red) following transfection with either scramble siRNA or YAP siRNA. Cells stained with Hoechst (blue) to mark all nuclei. Scale bar: 25 µm. Quantitation of the fluorescence intensity of BrdU has been represented on the right. C BrdU incorporation assay in TSCs following transfection with either empty vector backbone or YAP cDNA. D Photomicrographic images of BrdU incorporated cells (red) following transfection with either empty vector backbone or YAP cDNA. Cells were stained with Hoechst (blue) to mark all nuclei. Scale bar: 25 µm. Fluorescence intensity of BrdU has been quantified and represented on the right. Normalization was done against the cell number (CTCF/Cell) in three different biological replicates. E Coimmunoprecipitation of endogenous CDX2 with YAP using TSC lysate. Immunoprecipitation (IP) with anti-YAP antibody was followed by western blot with anti-CDX2 antibody. IP using an isotype matched antibody was used as a negative control. IgG heavy chain detected in each sample was used as loading control. F Quantitative real-time PCR analysis of Cyclin D1 transcripts in TSCs transfected with either scramble or YAP siRNA. G Western blot analysis of CYCLIN D1 using cell lysates from TSCs transfected with either scramble or YAP siRNA. GAPDH was used as an endogenous control. Densitometric analysis of the protein bands from G using NIH ImageJ is shown with the bar graphs on the right side of the blots. H Quantitative real-time PCR analysis of Cyclin D1 transcripts in TSCs transfected with vector back bone or YAP cDNA. I Western blot analysis of CYCLIN D1 using cell lysates from TSCs transfected with vector back bone or YAP cDNA. GAPDH was used as an endogenous control. Densitometric analysis of the protein bands from I using NIH ImageJ is shown with the bar graphs on the right side of the blots. Values are represented as mean ± SEM from three independent biological replicates. Statistical analysis was performed using Student’s unpaired t-test. *p < 0.05; **p < 0.005