Figure 4

(A) Effect of celecoxib and anti-VEGF on the proadhesive response of B16M cells to BMSC-CM in vitro. Murine B16M cells received 1 μg/ml celecoxib for 30 min and then incubated in the presence of basal medium, BMSC-CM, LPS-treated, BMSC-CM, rmVEGF (10 ng/ml) or PGE2 (100ng/ml) for 4 h. In some experiments, B16M cells received 1 μg/ml murine anti-VEGF monoclonal antibody 30 min prior to BM conditioned media. Once treatments were finished, a B16M adhesion assay to BMSCs was performed. In other experiments, anti-VCAM-1 antibody (10 μg/ml) was added to the cultures of BMSCs 30 min before adhesion assay. Differences were statistically significant cells (P < 0.01) with respect to (*) basal medium- or (**) BMSC-CM- or (+) LPS-treated BMSC-CM, (#) rmVEGF-treated melanoma cells or (&) PGE2-treated melanoma cells according by ANOVA and Bonferroni's post-hoc test. (B) Effects of LPS on TNFα and VEGF production. Supernatants were obtained from B16M cells incubated 1 ng/ml LPS for 6 h. A competitive enzyme immunoassay was carried out to determine murine TNFα and VEGF concentration. Statistical significance by ANOVA and Bonferroni's posthoc test (*) p < 0.01 vs untreated BMSC. (C) Effect of LPS on VCAM-1 expression by BMSC. BMSC were treated with basal medium and LPS (1 ng/ml) for 6 h. Then, cell lysates were collected and assayed for VCAM-1 and β-tubulin levels by western immunoblot. (D) Effect of celecoxib on TNFα-treated B16M cells. B16M cells received 1 μg/ml celecoxib 30 min prior to TNFα incubation for 4 h (10 ng/ml). Statistically significant by ANOVA and Bonferroni's posthoc test (*) p < 0.01 vs untreated B16M cells, (**) p < 0.01 vs TNFα-treated B16M cells. All data represent media ± SD of 3 separate experiments, each in six replicates (n = 18)