Figure 1
From: Exchange factors directly activated by cAMP mediate melanocortin 4 receptor-induced gene expression
Significant role for EPACs in α-MSH-induced CRE activation: HEK-293-MC4R cells.
(A) cAMP accumulation was measured after labeling of HEK-293-MC4R cells with [3H]-adenine followed by the purification of [3H]cAMP and [3H]ATP by sequential chromatography. Cells were stimulated with 1 μM α-MSH for 30 min at 37 °C (N = 5). Asterisks indicate a significant difference between MSH and basal using the two-sample Student’s t-test. In (B,C) HEK-293-MC4R cells were transfected with a reporter gene construct harboring the firefly luciferase gene under the control of a CRE-dependent promoter. In (B) cells were stimulated with various concentrations of α-MSH for 4 h. Means of 4 independent experiments performed in quadruplicates were compiled. Asterisks indicate a significant difference to cells stimulated with the lowest α-MSH concentration (−12) using one-way ANOVA followed by Dunnett’s analysis. In (C) cells were stimulated or not with 1 μM of α-MSH for 4 h after 30 min pretreatment with KT-5720 (5 μM; N = 7), A-812511 (10 μM; N = 8), rp-Br-cAMPs (50 μM; N = 6), ESI-09 (20 μM; N = 14), HJC-0197 (25 μM; N = 3) or ESI-05 (50 μM; N = 4) or the carrier DMSO (0.1% or 0.2%). Data were compiled first by normalizing α-MSH-induced CRE activation to the carrier or to the inhibitor alone and then by calculating the inhibition as percentage. Hashed signs indicate a significant difference to zero using the one-sample Student’s t-test.
