Figure 8. siRNA knockdown of the caveolin-1 protein expression and the correlation of phospho caveolin-1 protein expression level in cells incubated in serum, serum starved or serum starved and rLOX-PP treated cells.

(A) For confirmation of siRNA knockdown of the caveolin-1 protein, cells were transfected under similar conditions with either control (non-silencing) siRNA or siRNA directed against caveolin-1. Maximum reduction of caveolin-1 due to specific siRNA transfection was observed 75 hours post-transfection. Sixty hours after transfection, cells were serum starved for 12 hours followed by incubation with or without rLOX-PP for an additional 3 hours. Cells extracts were subjected to SDS PAGE. Cell extract samples were probed with phosphor-caveolin-1, total caveolin-1 and band intensity against beta actin was quantified. And relative phosphor-CAV-1 protein expression was determined. This experiment was performed at least three times with the same outcomes. Data are means +/− SD; n = 3; *, two-tailed p-value <0.0001. (B) MDA-MB-231(left) and SCC9 cells (right) were transfected with either CAV-1 siRNA or control siRNA. After transfection, cells were serum starved for 12 hours followed by incubation with or without rLOX-PP-Atto565 (red) for an additional 15 min on ice, and then incubated at 37° C for 15 minutes in the 5% CO₂ incubator. Cells then were fixed, permeabilized and stained for F-actin (green), DNA (blue) and total caveolin-1(magenta) or –phospho caveolin-1 (magenta). Merged images with rLOX-PP-Atto565 (above) and without rLOX-PP-Atto565 (below) treatment were reconstructed with Zen Black Edition software.