Fig. 3.

Silencing of α5β1 integrin delayed gefitinib-mediated EGFR endocytosis. a Left panel: U87 and U7α5- cell lysates were immunoblotted to detect EGFR, α5 integrin and GAPDH. Right panel: densitometric analysis. b EGF-Alexa488 internalization assays in U87 cells and U87α5-. Following serum-starvation and EGF-Alexa488 binding to the cell surface, cells were replaced in complete medium at 37 °C to allow internalization of the ligand in presence of gefitinib (20 µM) or DMSO. The internalization was measured by integrated fluorescence density on 20–30 cells/experiment of 3 independent experiments and reported in the histogram by arbitrary units of fluorescence (AUF). ***p < 0.001. c Confocal microscopy detection of actin filaments (green), EGFR (red) and EEA1 (cyan) in U87 and U87α5- cells treated with vehicle (control) or gefitinib (20 µM, 4 hours). High-magnification images are from the inserts into the peri-nuclear area. Scale bar = 20 μm. d EGFR/EEA1 colocalization using Mender’s coefficient. Confocal images from 3 independents experiments were analyzed with JACOPs plugin of ImageJ software