ERb Function in Mesothelioma Figure 1. ERb expression in ERs negative MMe cells reduces their growth rate. A) Western Blot analysis of cell extracts from mock- and ERb expressing MSTO-211H cells. Representative of three separate experiments. B) Upper panels show phase contrast microphotographs (200X magnification) of mock- or ERb-transfected MSTO-211H cells, visualizing the acquisition of a more epithelioid phenotype in transfected cells. Lower panels show cells fixed in ethanol and stained for actin with phalloidin–TRITC as described. Note the actin rearrangement in ERb expressing cells (400X magnification). C) Cell proliferation curves of mock- and ERb-transfected MSTO-211H cells cultured in complete medium for 24 and 48 hours. Each value represents mean 6 SD (n = 3). D) Total soft agar colony counts for mock- or ERb-transfected MSTO-211H cells were done by three independent investigators microscopically visualizing individual colonies (clusters of 15 or more cells) in 10 random microscopic fields. Columns represent the fold increase of the mean number of colonies in 10 fields; bars, SD; * p,0.05. Representative of three separate experiments. doi:10.1371/journal.pone.0014110.g001 evidence suggests that the EGFR-caveolin interaction leads to reduced activation of EGFR signaling [26]. The interaction between androgen receptor and EGFR in the caveolae of prostate cancer cells has been recently reported [27]. Consequently, we investigated the physical interaction between ERb, EGFR and caveolin-1 in REN cells. Firstly, membrane associated EGFR was immunoprecipitated from lysates of REN cells treated 1 and 5 minutes with 5 ng/ml of human recombinant EGF. As shown in Fig. 4A, Western blot analysis evidenced increased amounts of ERb and caveolin 1 in EGFR immunoprecipitates upon EGF stimuli. Membrane-associated EGFR was then immunoprecipi- tated from mock- and ERb -transfected REN cells that were treated for 5 minutes with EGF or left untreated, and then analyzed by immunoblotting with EGFR, ERb and caveolin-1 PLoS ONE | www.plosone.org 3 November 2010 | Volume 5 | Issue 11 | e14110