FIG 4.

WT-BEC survival and the Hippo pathway are unaffected by YM155 treatment. (A to K) Western blot analyses reveal that survivin (A), cytoplasmic P-YAP (B), nuclear YAP (C), CD31 (D), VE-cadherin (E), cleaved caspase 8 (CC8) (F), cleaved caspase 3 (CC3) (G), MMP2 (I), MMP9 (J), and PCNA (K) levels in WT-BEC were not affected in the presence of YM155 (0, 50, and 500 nM). In contrast, in EOMA cells, MMP9 was not affected in the presence of YM155 (J), and survivin (A) and MMP2 (I) expression levels were reduced in the presence of 500 nM YM155, while CD31 (D), VE-cadherin (E), cleaved caspase 3 (G), and cytoplasmic P-YAP (B) expression levels were increased and nuclear YAP (C) and PCNA (K) expression levels were decreased. Data represent the mean ratios of survivin (A), P-YAP (B), CD31 (D), VE-cadherin (E), MMP2 (I), MMP9 (J), and PCNA (K) to β-actin; YAP (C) to histone H1; and cleaved caspase 8 (F) and caspase 3 (G) to full-length caspase from triplicate samples for each cell type (WT-BEC and EOMA cells) with different concentrations of YM155 (0, 50, and 500 nM) ± SD (*, P < 0.05; **, P < 0.01; ***, P < 0.001). In panel H, numbers of apoptotic EOMA cells were significantly increased in the presence of YM155. Data are means ± SD from triplicate experiments (**, P < 0.01). (L) Phase-contrast and merged DAPI and survivin, DAPI and P-YAP, DAPI and YAP, DAPI and CD31, DAPI and VE-cadherin, and DAPI and cleaved caspase 3 immunofluorescence micrographs of WT-BEC treated with 0 nM and 500 nM YM155 confirming our Western blot analyses. Bar = 100 μm. (M) Morphological analysis of WT-BEC and EOMA cells with YM155 treatment (0, 50, and 500 nM) plated at low cell density (0 h) (top), just at confluence (48 h) (middle), and until overconfluent (120 h) (bottom), using Hoffman interference reflection microscopy. EOMA cells were contact inhibited in the presence of 500 nM YM155 and modestly inhibited at 50 nM YM155. WT-BEC morphology was not affected by YM155 treatment. Bar = 100 μm.