Fig. 5
EGFR is required for both LPS- and IL-10-stimulated macrophage-mediated gastric cancer cell invasion. a EGFR expression was transiently silenced by transfection with validated siRNA, at 48 h post-transfection. b Invasion assays with AGS cells (AGS), AGS cells with Lipofetamine2000 (AGS + Lipofect) or with Lipofectamine2000 transfected together with siRNA directed to EGFR (AGS + siRNA EGFR) were performed in the presence or absence of LPS- (LPSmac) or IL-10-stimulated (IL-10mac) macrophages. These assays were conducted at 24 h post-transfection and stopped at 48 h post-transfection, when maximum inhibition was achieved. Bars represent mean values of independent experiments performed with macrophages from at least 4 different donors and flags indicate standard error mean * significantly different from AGS at p < 0.05; ** significantly different at p < 0.05. c Tyrosine phosphorylation status of EGFR residue Y1086 (red) after 1 h of incubation of AGS cells with RPMI (CM RPMI) or CM from unstimulated (CMmac), LPS- (CM(LPSmac)) or IL-10-stimulated (CM(IL-10mac)) macrophages. Nuclei were counterstained with DAPI (blue). Scale bar represents 10 μm. The image is representative of independent experiments performed with CM of macrophages, from at least three different donors. d AGS or RKO cells were treated or not (RPMI), during 1 h, with CM from unstimulated (CMmac), LPS- (CM(LPSmac)) or IL-10-stimulated (CM(IL-10)mac) macrophages. Cell lysates were immunoblotted for phosphorylated and total EGFR (Y1086), c-Src (Y416), Akt (S473), ERK1/2 (T202/Y204), and p38 (Thr180/Tyr182). Immunoblots were analyzed by densitometry analysis in comparison with corresponding α-tubulin and total protein expression levels. Images are representative of independent experiments performed with CM of macrophages from at least 3 different donors