Figure 6. YAP1 WW domain interacts with Oct4 transcription factor.

(A) YAP1-Oct4 binding is detected by immunoprecipitation with an Oct4 antibody followed by western blot analysis with YAP1 in asynchronous A549 and H1650 cells. Normal IgG was used as a control for the IP reaction, and western blot for E2F was a negative control for Oct4 binding. (B) YAP1-Oct4 interaction is disrupted by a WW domain peptide. The presence of Oct4 in YAP1 immunoprecipitation was abolished by the WW domain peptide, but not a scrambled peptide, as seen by western blotting; binding of TEAD2 to YAP1 was not affected. Normal IgG is used as a control for the IP reaction. (C) An in vitro binding assay using 293T cell lysate overexpressing wild type YAP1 (YAP1 WT OE) or WW mutant YAP1 (YAP1 WW mutant OE) and GST-Oct4 showing that mutation in the WW domain of YAP1 abrogates its binding to Oct4. (D) Expression of YAP1 WW domain mutant lacks the ability to induce the Sox2 core promoter-luc activity alone or in the presence of Oct4 exogenous expression as shown in a luciferase assay. The increased Sox2 core promoter-luc activity is seen when YAP1 WT construct was used alone or in combination with Oct4. 1 μg of DNA is used for all the plasmid constructs in this assay (E) WW domain peptide conjugated to penetratin (WW peptide conjugate) inhibited the YAP1 mediated induction of Sox2 core promoter-luc activity, but a scrambled peptide conjugated to penetratin (Scrambled peptide conjugate) did not. (F) WW peptide conjugate, but not a scrambled peptide conjugate, abrogates angiogenic tubule formation by H1650 SP cells (G) SP cells from A549 and H1650 cell lines form less number of spheres when with WW peptide conjugate, but not scrambled peptide conjugate. The above data is represented as mean ± SD of three independent experiments. * represents p < 0.05, ** represents p < 0.01.