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 h post‐transfection. Sixty hours after transfection, cells were serum starved for 12 h followed by incubation with or without rLOX‐PP for an additional 3 h. 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 phospho‐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 h 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 min 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.