Reduction of E-cad by siRNA increased the levels of total EGFR and cell surface EGFR in both 686LN and PCI-37A cells. (a) Basal E-cad and EGFR expression in 4 SCCHN cell lines. (b) Both E-cad siRNA and non-targeted control siRNA were transfected using HiPerFect transfection reagent. The effects of E-cad reduction by siRNA transfection on EGFR expression at the protein level were evaluated by Western blot. At 72 hours after the transfection, EGFR protein levels were clearly increased in both 686LN and PCI-37A cells. (c). To quantify EGFR expression on the cell membrane, flow cytometric analysis of cell surface staining was performed. The cells transfected with siRNA (48 hrs after transfection) were incubated with monoclonal mouse-anti-EGFR antibody (Santa Cruz). The level of EGFR protein on the cell surface was examined by flow cytometry analysis using FACScan (BD). Data were analyzed by the software FlowJo (Ashland, OH). Data indicate that in both 686LN and PCI-37A cells, the level of cell surface EGFR protein was significantly increased upon reduction of E-cad based on three measurements (p < 0.001 for both cell lines). Error bars represent standard deviation.