Fig. 7

The RAMP1-Gαi3-PKA axis regulates YAP mediated by CREB. In the OE+KG-501 group, RAMP1 OE cells were first treated with KG-501 (10 μM) for 12 h and then with KG-501 (10 μM) and DOX (5 μg/ml) for 48 h. In the OE and VEC groups, RAMP1 OE and VECs were first treated with DMSO (10 μM) for 12 h and then with  DMSO (10 μM) and DOX (5 μg/ml) for 48 h. A Confocal immunofluorescence of PCNA (red), Actin (green) and DAPI (blue). Scale bar = 20 μm. B Dot plots show quantification of the frequency of PCNA-positive cells in each field of view. C Western blot analysis of PCNA, CREB, pCREB (S133), YAP and pYAP (S127). Total protein was stained as an endogenous control. D Quantification of the protein band intensities in C. The data were normalized to LNF. E Cell proliferation ability measured by confluence measurements normalized to hour 0 and calculated using IncuCyte (Essen BioScience). F qPCR analysis of the YAP mRNA levels. The data were normalized to the amount of β-actin mRNA. G Western blot analysis of the cytoplasmic and nuclear fractions of YAP and CREB. Total protein was stained as an endogenous control. H Quantification of the protein band intensities of G. 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. I Confocal immunofluorescence of CREB (red), Actin (green) and DAPI (blue). Scale bar = 20 μm. J Confocal immunofluorescence of YAP (red), Actin (green) and DAPI (blue). Scale bar = 20 μm. K DNA agarose gel electrophoresis of YAP promoter DNA quantification using the CUT&RUN assay and qPCR with different primers. L Dual-luciferase reporter assay of the CBS wild-type (WT) YAP promoter and mutated (Mut) YAP promoter. The experiments were performed in triplicate. The data were presented as means ± SD, and significant differences were evaluated using unpaired t test. *P < 0.05, **P < 0.01, ***P < 0.005 and ns, not significant