Fig. 3

Cholesterol depletion partially delocalizes cortactin from the perinuclear region but does not affect its phosphorylation state. Cell lines were either serum-starved combined or not with 2 mM mβCD (chol depletion) for 2 h then stimulated for 4 h with serum-containing medium or serum-starved for 2 h then incubated in 10 µM GM6001-supplemented medium (MMP inhibition). All conditions were tested for cortactin distribution by confocal microscopy (A) and expression and activation by Western blots (B–D). A Confocal images of cells plated on fibronectin-coated coverslips, serum-starved or treated with mβCD or GM6001. Cells were (immuno) labeled with anti-Cortactin, Phalloidin (F-actin) and Hoechst (nuclei). Blue areas, perinuclear regions occupied by cortactin. White areas and corresponding insets, cortactin at the cell edge. White arrowheads, cell edge protrusions (n = 1–3). B Western blots showing the expression levels of cortactin and phosphorylated cortactin (pCortactin Y421) with both isomers at 80 kDa and 85 kDa in the 6 most invasive cell lines treated of not with mβCD or GM6001. GAPDH was used as loading control (n = 2–3). C, D Quantification of total cortactin (80 + 85 kDa forms) protein abundance (C) and phosphorylation level of the 85 kDa form (ratio of pCortactinY421/cortactin; D). Data are expressed as percentage of the untreated control (n = 2–3)