FIG 5.

Expression of CCN1 controls YAP cellular localization and activity in HRECs. (A) Expression of YAP and pYAP in HRECs expressing increasing concentrations of Ad-CCN1 (MOI, 1 to 5), as determined by Western blotting and densitometric scanning of protein bands. (B) Cytoplasmic localization of YAP in HRECs overexpressing CCN1. Cells were transduced with either Ad-luc or Ad-CCN1 (3 MOI) and further transfected with GFP-tagged YAP (i.e., pEGFP-C3-hYAP1). (C and D) Expression of CCN1, YAP, and pYAP in HRECs transduced with Ad-CCN1 and exposed to VEGF (50 ng/ml) for 4 h. *, P < 0.0003 (n = 3). (E) Effects of CCN1 on YAP transcriptional activity, as determined upon cell transfection with the 8×GTIIC-lux plasmid. *, P < 0.001 (n = 3). (F) Effect of function-blocking integrin antibodies (Ab) on CCN1-induced YAP phosphorylation. *, P < 0.0004; **, P < 0.0003 (n = 3). (G and H) Effects of small Rho GTPases and pharmacological inhibitors on CCN1-mediated YAP inactivation. *, P < 0.05 versus conditions with VEGF (n = 3); NS, not significant. (I) Phosphorylation status of potential YAP kinases (e.g., Erk1/2, Akt, and Src kinase) upon VEGF stimulation of CCN1-overexpressing cells. Protein lysates were analyzed by Western immunoblotting with the indicated antibodies. Cntl, control; Veh, vehicle; pYAP, pErk1/2, pAkt, and pSrc kinase, phosphorylated YAP, Erk1/2, Akt, and Src kinase, respectively.