Fig. 1.
Epac increases melanoma cell migration. A: Epac increases melanoma cell migration. Cells were infected with adenovirus harboring LacZ, Epac1, Epac2, and protein kinase A (PKA) followed by the migration assay in the presence or absence of 50 μM 8-(4-methoxyphenylthio)-2'-O-methyladenosine-3',5'-cAMP (8-pMeOPT) or 50 μM N6-monobutyryl-cAMP (6-MB-cAMP). Both 8-pMeOPT and Epac overexpression, but neither 6-MB-cAMP nor PKA overexpression, increased melanoma cell migration. *P < 0.01 vs. LacZ. n = 4. B: basal migration assay in melanocytes and melanoma cell lines is shown. Basal migration was higher in melanoma cell lines than in melanocytes and in SK-Mel-24 cells than in SK-Mel-2 cells. n = 4. C: immunoblots for Epac1 and Epac2. Epac1 protein expression was more abundant in melanoma cell lines than in melanocytes and in SK-Mel-24 cells than in SK-Mel-2 cells. Epac2 protein expression was more abundant in melanocytes than in melanoma cell lines. Bar graphs show densitometric analyses of the blots. n = 4. D: quantitative real-time PCR (qPCR) showed that Epac1 small interfering RNA (siRNA) decreased mRNA expression in SK-Mel-2 cells or SK-Mel-24 cells are shown. n = 4. E: immunoblots showed that Epac1 siRNA decreased Epac1 protein expression in both SK-Mel-2 and SK-Mel-24 cells. F: Epac1 siRNA decreased basal cell migration in SK-Mel-2 cells or SK-Mel-24 cells. *P < 0.01 vs. control siRNA. n = 4. G: video-recorded analysis showed that SK-Mel-2 cells overexpressing Epac1 significantly increased cell motility. n = 10. Supplementary video files; video-recorded cell motility is shown. 1 second in video approximately corresponds to 1 h recording. H: invasion assay showed that both 8-pMeOPT and Epac1 overexpression increased invasion of SK-Mel-2 cells. n = 4. I: immunoblot showed that 6-MB-cAMP, but not 8-pMeOPT, increased phosphorylation of cAMP-response element-binding protein (CREB). J: immunoblot showed overexpression of Epac1, Epac2, and PKA with adenoviral infection in SK-Mel-2.