Endothelial CCL2 regulates adrenomedullin expression and release in tumor cells in vitro. (a–d) HUVECs were transfected with scrambled control siRNA or siRNA directed against GNAS and/or CCL2 and were cultured with GFP-MeWo for the indicated time periods. Endothelial cell proliferation was determined by staining for Ki67 (a), and the number of GFP-expressing tumor cells was determined by immunofluorescence (b) or determined as the protein level of GFP by Western blot analysis (c). In addition, expression of ADM was analyzed by immunoblotting (d). The bar diagrams show the statistical evaluation (a and b; n = 3 independent experiments) or the relative densitometric values based on Fiji software (c and d; n = 3 independent experiments). (e) HUVECs were transfected with control siRNA or siRNA directed against GNAS and cells were co-cultured for 24 h with MeWo cells transfected with control siRNA or siRNA directed against CCR2. Thereafter, the expression level of ADM was determined by Western blot analysis. The bar diagrams represent the relative densitometric values of the band recognized by the anti-ADM antibody based on Fiji software (n = 3 independent experiments). Bar length: 100 μm (a and b). Data represent mean values ± SEM; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001 (two-way ANOVA and Bonferroni’s post hoc test [c and e], and one-way ANOVA and Tukey’s post hoc test [a, b, and d]).