Fig. 5

The PKA inhibitor H-89 reduces CREB and YAP levels and inhibits MSF proliferation. In the OE+H-89 group, RAMP1 OE cells were first treated with H-89 (1 μM) alone for 24 h and then with H-89 (1 μM) and DOX (5 μg/ml) for 48 h. In the OE and VEC groups, RAMP1 OE and VECs were first treated with DMSO (1 μM) alone for 24 h and then with DMSO (1 μM) and DOX (5 μg/ml) for 48 h. A Western blot analysis of PCNA, PKA, CREB, pCREB (S133), YAP and pYAP (S127). Total protein was stained as an endogenous control. B Quantification of the protein band intensities in A. The data were normalized to LNF. C qPCR analysis of the YAP mRNA levels. The data were normalized to the amount of β-actin mRNA. D Cell proliferation ability measured by confluence measurements normalized to hour 0 and calculated using IncuCyte (Essen BioScience). E Western blot analysis of the cytoplasmic and nuclear fractions of YAP and CREB. Total protein was stained as an endogenous control. F Quantification of the protein band intensities of E. The data were normalized to LNF. C-CREB means cytoplasmic CREB, N-CREB means nuclear CREB, C-YAP means cytoplasmic YAP, and N-YAP means nuclear YAP. G Confocal immunofluorescence of CREB (red), Actin (green) and DAPI (blue). Scale bar = 20 μm. H Confocal immunofluorescence of YAP (red), Actin (green) and DAPI (blue). Scale bar = 20 μm. The experiments were performed in triplicate. The data are presented as means ± SD, and significant differences were evaluated using unpaired t test. *P < 0.05, **P < 0.01 and ***P < 0.005