CCL2 promotes chemotaxis and migration of endothelial cells in vitro. (A) HUVECs were stained with an anti-CCR2 antibody (blue histogram) or isotype control (red histogram) and analyzed by FACS. (B) Migration of HUVECs toward conditioned medium of neoplastic MC (CM) was determined using a modified Boyden chamber assay. CM was pre-incubated with an antibody against CCL2 or an isotype control antibody (αISO) to determine the specificity of the observed effect. The number of migrated cells per field was counted and normalized to control level. (C-E) Migration of HUVECs in a scratch assay. The upper panel of (C) shows the scratch before and the lower panel after 24 hours of incubation with CM. CM was pre-incubated with an antibody against CCL2 or an isotype control antibody (αISO) as described above. The number of cells in the scratch was counted and normalized to control level. (D) Respective numbers of mean ± SD of at least 3 independent experiments. (E) In similar experiments, conditioned medium from neoplastic MCs after transduction with 2 different short hairpin RNAs (shRNAs) targeting CCL2 (#1 and #2) or an NTC was used. Results represent the mean ± SD of at least 3 independent experiments. *P < .05, **P < .01, ***P < .001.