Figure 4

Lipid raft disruption reduces uPAR and MMP-9 activity in lipid raft fractions of MDA-MB-231 and ZR 751 cells. (A) & (B) Characterization of lipid raft fractions. (A) MDA-MB-231 and (B) ZR 751 cells were left untreated or pretreated with 10 mM MβCD for one hour. Cells were washed, lysed, and raft and non-raft fractions were collected as described earlier. Fractions were characterized as raft fractions or non-raft fractions and western blotted for caveolin or flotillin (raft marker) and transferrin (non-raft marker). Fractions 3 to 5 were pooled as raft fractions, and pooled fractions 6 to 8 were taken as non-raft fractions. (C) & (D) Effect of lipid raft disruption on uPAR and MMP-9 gene expression in breast carcinoma cells. (C) MDA-MB-231 and (D) ZR 751 cells were left untreated or pretreated with different concentrations of MβCD for one hour, washed, complete medium added, and total RNA isolated at 8 h using the TRIzol method. 50 ng of DNAse-treated RNA/μL were subjected to semi quantitative RT-PCR with primers specific for uPAR and MMP-9. Expression of GAPDH was used to verify equal loading of cDNA. (D) Densitometric analysis of uPAR and MMP-9 expression at the mRNA level (mean ± SE, n = 3) (*p < 0.001, **p < 0.0001).