Fig. 4

Interference with Gαi3 after RAMP1 overexpression decreases PKA, CREB, and YAP expression and MSF proliferation. RAMP1 OE cells were reverse-transfected with 50 nM of small interfering RNAs (siRNAs) specifically targeting Gαi3 (siGαi3-1 and siGαi3-2) or a negative control sequence (siNC) for 48 h and then were treated with DOX (5 μg/ml) for 48 h. A Western blot analysis of Gαi3. Total protein was stained as an endogenous control. B Quantification of the protein band intensities of A. The data were normalized to LNF. C qPCR analysis of the Gαi3 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 calculated using IncuCyte (Essen BioScience). E qPCR analysis of the YAP mRNA levels. The data were normalized to the amount of β-actin mRNA. F Western blot analysis of PCNA, PKA, CREB, pCREB (S133), YAP, and pYAP (S127). Total protein was stained as an endogenous control. G Quantification of the protein band intensities in F. The data were normalized to LNF. H Confocal immunofluorescence of PCNA (red), Actin (green) and DAPI (blue). Scale bar = 20 μm. I Dot plots show quantification of the frequency of PCNA-positive cells in each field of view. J Western blot analysis of the cytoplasmic and nuclear fractions of CREB and YAP. Total protein was stained as an endogenous control. K Quantification of the protein band intensities of J. 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. L Confocal immunofluorescence of CREB (red), Actin (green) and DAPI (blue). Scale bar = 20 μm. M 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, ***P < 0.005 and ns, not significant