Figure 3

Significant role for EPACs in α-MSH-induced CRE activation: mHypoA-2/10-CRE cells.

(A) Cells were stimulated with α-MSH for 30 min (N = 5). Asterisks indicate a significant difference using the two-sample Student’s t-test. (B) Cells were stimulated wit α-MSH or co-stimulated with HS-024 for 4 hours (N = 3). Asterisks indicate a significant difference using one-way ANOVA followed by Dunnett’s analysis. (C) Cells were pretreated or not with neuropeptide Y (NPY) and then stimulated with α-MSH. Data were compiled by calculating the x-fold over basal (N = 5). Asterisk indicates a significant difference using the two-sample Student’s t-test. (D) Cells were stimulated with increasing concentration of α-MSH or γ-MSH. Means of 11 (α-MSH) or 3 (γ-MSH) experiments performed in triplicates were compiled. Asterisks indicate a significant difference to cells stimulated with the lowest concentration using one-way ANOVA followed by Dunnett’s analysis. (E) Cells were stimulated or not with 1 μM of α-MSH for 4 h after 30 min pretreatment with KT-5720 (5 μM; N = 6), A-812511 (10 μM; N = 11), rp-Br-cAMPs (50 μM; N = 5), ESI-09 (20 μM; N = 14), HJC-0197 (25 μM; N = 5) or ESI-05 (50 μM; N = 10) or the carrier DMSO. Data were compiled 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 using the one-sample Student’s t-test. (F) Cells were preincubated with 0.2% DMSO or 20 μM ESI-09 and in (G) with 0.1% DMSO or 25 μM HJC-0197 for 30 min and then stimulated with 20% serum for 4 hours. Asterisks indicate a significant difference using two-way ANOVA followed by Bonferroni analysis. (H) Cells transfected with a specific Rap-1a or a control siRNA were subjected to western-blot analysis. In (I) α-MSH- (1 μM) or serum-induced (20%) CRE activation was determined in cells either transfected with 50 nM of a specific Rap-1a or a control siRNA. Data were compiled first normalized to control cells or cells transfected with the Rap-1a siRNA and then by calculating the inhibition as percentage (N = 5). Hashed signs indicate a significant difference using the one-sample Student’s t-test, asterisks using the two-sample Student’s t-test.