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. 2018 Mar 20;8:4904. doi: 10.1038/s41598-018-23199-4

Figure 6.

Figure 6

Expression of active CRTC2/3 inhibits VSMC proliferation but not migration, and CRTC2/3 contribute to cAMP-induced inhibition of proliferation. Asynchronously proliferating cells in 5% FCS were infected with control adenovirus (Ad:control) or adenovirus expressing active-CRTC2 (Ad:CRTC2) or active-CRTC3 (Ad:CRTC3) for 24 h and incubated for a further 6 h with 10 µM BrdU to label proliferating cells (A, n = 4). VSMC expressing active CRTC2 or CRTC3 were serum starved for 24 h and proliferation was stimulated with 5% FCS (B, n = 5) or 25 ng/ml PDGFBB (C, n = 7) for 24 h, with BdrU present during the final 6 h. Serum-starved VSMC were stimulated with 2 µM HG-9-91-01 for 45 min and nuclear translocation of endogenous CRTC2 and CRTC3 was analysed by cell fractionation and western blotting (D). VSMC were transfected with CREB-LUC and serum starved for 18 hours ± 2 µM HG-9-91-01. CREB-LUC was quantified in cell lysates (E). Asynchronously proliferating cells were stimulated with 2 µM HG-9-91-01 for 24 hours with BrdU present for the final 6 hours (F). VSMC were transfected with scrambled siRNA (siNEG) or siRNA against CRTC2 and CRTC3 (siCRTC2/3) and grown in 5% FCS for 24 h. Cells were treated with 1 µg/ml BAY60-6583 (BAY) for 24 h with BrdU present for the final 6 h to label cells (G, n = 4). VSMC were transfected with siNEG or siRNA against CREB. Cells were then infected with Ad:control or Ad:CRTC2. Proliferation was quantified 24 hours later by labelling with BrdU for 6 hours (n = 4; H).