Downmodulation of CD146 expression and migration abilities of the MDA-MB-231 cell line. (a) CD146 mRNA expression in the MDA-MB-231 cell line 72 hours after transfection with siRNAs targeting CD146. Two different siRNAs (si78 and si79) and two controls (si78mut and siGFP) were used. CD146mRNA expression was normalized to GAPDH and expressed relatively to the native cells (arbitrarily 100%). (b) Protein expression (PE) measured by flow cytometry, one representative experiment. (c) Chemotactic migration evaluated after 24 hours, using uncoated Boyden chambers and 10% FCS as chemoattractant. (d) Transmigration through the established human endothelial HBMEC cell line. (e) and (f) Wound healing assay. (e) Wound healing repair was evaluated after 24 hours; percentage of the initial wound surface repaired after 24 hours was estimated using ImageJ software. (f) A representative experiment of wound healing: MDA-MB-231 cells were transfected with siRNAs and, 3 days after transfection, a cell-free area (wound) was created in confluent cultures (t = 0); cells were allowed to migrate for 24 hours (t = 24) before image analysis. For (c), (d) and (e) results are expressed relative to the results obtained with the mutated si78 RNA (arbitrarily 100%) and represent the mean ± standard error of the mean of four independent experiments (except for (d), which represents two experiments).