FIGURE 3.

YAP depended on CD166. A and B, knockdown of CD166 (infected with CD166-sh1 (A) and CD166-sh2 (B), respectively) reduced YAP as well as its target gene expression as measured by Western blotting. C, overexpression of CD166 induced BIRC5 expression. Shown are Western blots of proteins as indicated in control (transfected with empty vector) and Bel-7402 cells with CD166 overexpressed. D, CD166 dose-dependently induced YAP expression as measured by Western blotting in HL-7702 cells. E, YAP did not affect CD166 expression. Shown are Western blots of CD166 and YAP in Bel-7402 and SMMC-7721 cells with or without YAP knocked down (infected with shRNA against YAP). F, endogenous expression patterns of YAP and CD166 in Bel-7402 and SMMC-7721 cells. G, high density culture of cells induced down-regulation of YAP and CD166 as measured by Western blotting in Huh7 and HepG2 cells. H, increased cell density reduced p-AKT substrate level. Western blots of proteins as indicated in lower or higher density cultured Huh7 cells. I, inhibition of PI3K/AKT inhibited both YAP and CD166 expression. Shown are Western blots of YAP and CD166 in Bel-7402 and SMMC-7721 cells treated with DMSO, LY294002 (20 μm), or wortmannin (50 μm) for 24 h. S, shorter exposure; L, longer exposure. J, knockdown of CD166 reduced YAP activity. Luciferase activities from the pUAS-LUC/TEAD-Gal4 system were measured in control cells (infected with GFP shRNA) and cells with CD166 knocked down (infected with CD166-sh1). K, CD166 facilitated YAP/TEAD interaction. In CD166-HA-transfected Bel-7402 cells, YAP was immunoprecipitated with an anti-FLAG antibody, and Western blotting analysis of TEAD was done by an anti-Myc antibody. L, YAP intracellular localization in Bel-7402 cells with or without CD166-HA overexpressed. Scale bar, 15 μm. M, no direct association between YAP and CD166. CD166-HA was co-transfected with YAP-FLAG into Bel-7402 cells as indicated. YAP and CD166 associations were examined by co-IP as indicated. Error bars, S.D.