Hepatocyte growth factor-mediated cell survival requires epidermal growth factor receptor expression. SUM102 cells were transduced with lentivirus expressing a non-silencing control or four nonoverlapping epidermal growth factor receptor (EGFR) small hairpin RNA (shRNA) targeting sequences. In addition, SUM102 cells were treated with gefitinib (gef) or hepatocyte growth factor (HGF), or were transfected with pcDNA3 or KD-EGFRmt as indicated. (A) Four days after transduction and/or transfection, cells were lysed, and lysates were used to determine expression levels of EGFR via immunoblotting. β-actin was used as a loading control. (B) Duplicate cells were trypsinized and replated in a 96-well plate. One series of gefitinib-treated cells were used as a control to demonstrate HGF protection from gefitinib treatment (far-left bar). Remaining cells were treated with or without HGF at 50 ng/ml without gefitinib for 72 hours. In addition, cells were transfected with KD-EGFRmt at the same time as transduction with shRNA (far-right bar). Cell viability was measured using MTS assays. Numbers under the immunoblot indicate relative densitometry. Lines indicate comparison samples. Each experiment was performed in triplicate at least three independent times. *Gef ± HGF, P < 0.001; -HGF ± EGFR knockdown, P = 0.001. N.S., not significant; NV, no virus; NS, nonsilencing. #1, #2, #3, and #4, arbitrary shRNA clone numbers. KD-EGFR, kinase-dead EGFR; EGFR*, mutated sequence within the shRNA targeting region.