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

Figure 5.

Figure 5

CRTC2 or CRTC3 mediate cAMP induced CREB activity. VSMC were infected with control adenovirus (Ad:control) or adenovirus expressing active-CRTC2 (Ad:CRTC2) or active-CRTC3 (Ad:CRTC3) and equal expression verified by western blotting with an anti-FLAG antibody (A, n = 4). Cells were transfected with CREB-LUC and 24 h later infected with CRTC or control adenovirus, luciferase activity was measured in cell lysates after 24 h (B, n = 4). mRNA levels of NR4A, CREM, SIK and HAS were assessed in Ad:CRTC2 or 3 infected cells by RT-qPCR (C, n = 4), and cell viability determined using trypan blue after 48 h viral expression (D, n = 4). VSMC were transfected with GAL4-NLUC together with either a GAL4, GAL4-CREBwild-type or GAL4-CREBS133A expression vector. Cells were infected with adenoviruses expressing constitutively active CRTC2 or CRTC3 and GAL4-NLUC activity quantified 24 hours later (E). VSMC were co-transfected with CREB-LUC reporter and scrambled siRNA (siNEG) or siRNA against CRTC2 and CRTC3 (siCRTC2/3). After 48 hours, cells were serum starved for 4 h before being treated with forskolin (FSK, 0.5 µM; n = 3), BAY60-6583 (BAY, 1 µg/ml; n = 3) or Cicaprost (CICA, 1 µM; n = 3) for 4 h. Cells were lysed for luciferase measurement (F). Data were analysed with one-way ANOVA with Student Newman Keuls post-tests; **indicates p < 0.01 relative to control, ##indicates p < 0.01 relative to siNEG.