Journal: International Journal of Molecular Sciences

Article Title: Prostaglandin E2 Exposure Disrupts E-Cadherin/Caveolin-1-Mediated Tumor Suppression to Favor Caveolin-1-Enhanced Migration, Invasion, and Metastasis in Melanoma Models

doi: 10.3390/ijms242316947

Figure Lengend Snippet: PGE2 increases migration and invasion of A375M (CAV1/E-cad) cells. A375 (Mock) and A375 (CAV1) cell lines cultured in the presence of IPTG were transiently transfected with pBATEM for 48 h to express E-cad. A375 (Mock), A375 (CAV1), A375 (E-cad), and A375 (CAV1/E-cad) cells were treated with 50 μM of PGE2 for 5 h. Then, the extracts were either prepared for immunoprecipitation experiments or cell migration and invasion measurements in transwell and matrigel assays, respectively. ( A ) CAV1 was immunoprecipitated from protein extracts using a polyclonal antibody. Immunoprecipitated CAV1 and co-immunoprecipitated E-cad were detected via Western blotting. ( B ) Representative Western blot of immunoprecipitates obtained in three independent experiments. E-cad and CAV1 protein levels were quantified in several experiments via densitometry and normalized to actin (mean ± SD, n = 3; ** p < 0.01). A375 (Mock), A375 (CAV1), A375 (E-cad), and A375 (E-cad/CAV1) cells were serum-deprived for 5 h and treated with PGE2 (50 μM) for 2 h. Then, CAV1 was immunoprecipitated from the protein extracts using a polyclonal antibody. Immunoprecipitated CAV1 and co-immunoprecipitated E-cad were detected via Western blotting. ( B ) Graph showing the CAV1 as normalized pixels (mean ± SEM) for each condition; n = 3; and ** p < 0.01 with respect to A375 (mock). ( C ) Graph showing E-cad as normalized pixels (mean ± SEM) for each condition; n = 3; ** p < 0.01 with respect to A375 (mock). ( D ) CAV1/E-cad as normalized pixels (mean ± SEM) for each condition; n = 3; and * p < 0.05 or ** p < 0.01 with respect to A375 (mock). ( E ) A375 (Mock), A375 (CAV1), A375 (E-cad), and A375 (E-cad/CAV1) cells were treated with 50 μM of PGE2 for 5 h. Then, 150.000 cells were seeded into Boyden chambers (transwells) in the presence of 50 μM PGE2. Migration was measured after 2 h. Significant differences are shown (* p < 0.05; n = 3). ( F ) Panels showing representative images of results obtained for cells migrating either without treatment (basal condition, upper panel ) or when treated with 50 μM of PGE2 for 5 h ( lower panel ), in both cases stained with crystal violet. ( H ) Cells cultured in the presence or absence of 50 µM of PGE2 for 5 h were used for the invasion assays. Cells (150.000) were seeded into matrigel Boyden chambers in the presence of 50 μM of PGE2. Invasion was measured after 48 h. Graphs show averages (mean ± SEM; n = 3). Significant differences are indicated (* p < 0.05). ( G ) Panel showing representative images obtained in invasion assays for cells cultured in the absence (basal condition, upper panel ) or presence ( lower panel ) of 50 μM of PGE2, in both cases stained with DAPI Bar = 50 μm. (* p < 0.05; ** p < 0.01; *** p < 0.001; n = 3).

Article Snippet: B16F10 metastatic murine melanoma cells (ATCC, #CRL6475, provided by Laurence Zitvogel, Institut Gustav Roussy, Villejuif, France) and the human melanoma cell line A375M (ATCC, #CRL1619), also employed previously [ ], were maintained in RPMI 1640 medium.

Techniques: Migration, Cell Culture, Transfection, Immunoprecipitation, Western Blot, Staining