ERb Function in Mesothelioma Figure 5. ERb expression influences response of MMe cells to Gefitinib. A) Effects of Gefitinib on viable number were evaluated in mock-, ERb- and shERb-transfected REN and in mock- and ERb- transfected MSTO-211H cell lines. Cells were incubated in serum-containing medium in the presence of 5 mM Gefitinib for 24–48 hours. As control 0.1% DMSO vehicle alone was used. Results are expressed as number of viable cells relative to control at 48 hours of treatment; bars, 6 SD; * p,0.05. Data are representative of three separate experiments. B) shERb-transfected REN and MSTO- 211H cells were treated with 5 ng/ml of EGF for 5 minutes in the absence or presence of 5 mM Gefitinib and detergent extracted. Levels of phosphorylated EGFR, ERK 1/2 MAP kinases and Akt were analyzed by immunoblot. Membranes were also blotted with antibodies to EGFR, Erk1/2, and Akt to evaluate protein expression. Tubulin was blotted to show equal amount of loading. Western blot analysis with anti ERb antibodies documents expression in transfected cells. Representative of three separate experiments. doi:10.1371/journal.pone.0014110.g005 PLoS ONE | www.plosone.org 8 November 2010 | Volume 5 | Issue 11 | e14110