Skip to main content
. 2020 Dec 2;40(49):9533–9546. doi: 10.1523/JNEUROSCI.0407-20.2020

Figure 4.

Figure 4.

Forskolin enhances CRTC2 activity and VEGF-A/VEGF-R2 promoter activity. A, Transient assay of bEnd.3 cells cotransfected with adeno-GAL4-fusion CRTC1 or CRTC2 and the adeno-5×GAL4-luciferase reporter with the internal reporter Adeno-TK-rluc. The specific transfection activities of either CRTC1 or CRTC2 were expressed as the fold activation of the empty adeno-GAL4 vector. Effect of KCl, Forskolin and Forskolin +KCl on GAL4 luciferase reporter activity are shown. B, Transient assay of bEnd.3 cells expressing the VEGF-A promoter (1853 bp) luciferase-adenovirus. Effects of KCl or Forskolin on luciferase reporter activity are shown. C, The bEnd.3 cells were cotransfected with adeno-GAL4-fusion CREB, CREB b-ZIP (–), CRTC2, or p300 and the adeno-5×GAL4-luciferase reporter with the internal reporter Adeno-TK-rluc. The bEnd.3 cells were treated with forskolin, dibutyryl-cAMP, or DMSO for 6 h, and a luciferase assay performed. D, The bEnd.3 cells were transiently transfected with the VEGF-A(−1853), VEGF-R1(−1160), VEGF-R2(−220), VEGF-R2(−780), VEGF-R3(−870)-luciferase adenovirus, and ad-TK-rluc. At 48 h postinfection, cells were exposed to forskolin, and after 12 h, a luciferase assay was performed. E, The bEnd.3 cells were transfected with the each EGFP, CRTC2WT, CRTC2-S307G, VP16-CREB, CBP, p300 S89A adenovirus vector and VEGF-A promoter (1853 bp) luciferase adenovirus. After 72 h, a luciferase assay was done. F, The bEnd.3 cells were cotransfected with adenovirus expressing the LacZ-, CRTC2-S171A, CRTC2-S171A/S275A double mutant, or CRTC2-S171A/S275A/S307A triple mutant, with the VEGF-A promoter (1853 bp) luciferase adenovirus. At 72 h postinfection, a luciferase assay was performed. Error bars represent mean ± SD. G, The bEnd.3 cells were transfected with the each EGFP, CRTC2WT, CRTC2-S171A, and CRTC2-S307G adenovirus vector and VEGF-R2 (KDR; −780)-luciferase adenovirus. After 72 h, a luciferase assay was done. Luciferase assay data are presented as mean ± SD. For A–G, *p < 0.05 by one-way ANOVA, followed by Tukey−Kramer post hoc analysis.