The expression of CSPG4 in aggressive breast cancer cell lines contributes to P-selectin binding. A) CSPG4 mRNA was measured by qRT-PCR and normalized to 18S values and log transformed. Means and standard deviations are shown. Comparisons were made by ANOVA and post-hoc analysis. B) Cell surface expression of CSPG4 was examined in the indicated breast cancer cell lines by flow cytometry using anti-CSPG4 225.28 mAb (10 μg/ml). Open histogram shows binding of the secondary antibody only, while filled histogram shows anti-CSPG4 225.28 mAb binding. One representative experiment out of three is shown. C) Expression of CSPG4 was inhibited by transient transfection of MDA-MB-231 cells with CSPG4 siRNA that led to a decrease in the binding of P-selectin (D) and anti-CS-A (E) to transfected cells In C, D and E the filled histograms show the binding of secondary antibodies only (control), the open histograms with solid lines show binding to vehicle-treated cells while the open histograms with dotted lines show binding to the siRNA-transfected cells.