Fig. 3

RAMP1 overexpression promotes proliferation by increasing YAP protein levels. In the OE+VP group, RAMP1 OE cells were first treated with veterporfin (VP, 0.01 μM) alone for 12 h and then with VP (0.01 μM) and DOX (5 μg/ml) for 48 h. In the OE and VEC groups, RAMP1 OE and VECs were first treated with DMSO (0.01 μM) alone for 12 h and then with DMSO (0.01 μM) and DOX (5 μg/ml) for 48 h. A Cell proliferation ability measured by confluence measurements normalized to hour 0  and calculated using IncuCyte (Essen BioScience). B Western blot analysis of PCNA and YAP. Total protein was stained as an endogenous control. C Quantification of the protein band intensities in B. The data were normalized to LNF. D qPCR analysis of the YAP mRNA levels. The data were normalized to the amount of β-actin mRNA. E Confocal immunofluorescence of PCNA (red), Actin (green) and DAPI (blue). Scale bar = 20 μm. F The dot plots show quantification of the frequency of PCNA-positive cells in each field of view. G Western blot analysis of the cytoplasmic and nuclear fractions of YAP. Total protein was stained as an endogenous control. H Quantification of the protein band intensities of G. The data were normalized to LNF. C-YAP means cytoplasmic YAP, and N-YAP means nuclear YAP. I Confocal immunofluorescence of YAP (red), Actin (green) and DAPI (blue). Scale bar = 20 μm. All 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