ERb Function in Mesothelioma Figure 2. ERb silencing promotes MMe cell proliferation. A) Western Blot analysis of cell extracts from mock- and ERb silenced REN cells. Representative of three separate experiments. B) Upper panels show phase contrast microphotographs (200X magnification) of mock- or shERb- transfected REN cells, visualizing the loss of contact inhibition and formation of foci in vitro. Lower panels show cells fixed in ethanol and stained with phalloidin-TRITC to stain for actin as described. Note the actin rearrangement in ERb silenced cells (400X magnification). C) Cell proliferation curves of mock- and shERb-transfected REN 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 shERb-transfected REN 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; bars, SD; * p,0.05. Representative of three separate experiments. doi:10.1371/journal.pone.0014110.g002 antibodies. ERb and caveolin-1 co-immunoprecipitated with EGFR to a minor extent in untreated cells however, EGF treatment promoted the interaction and more ERb and caveolin-1 was immunoprecipitated with EGFR. Over-expression of ERb resulted in an increased and EGF-independent association of these proteins (Fig 4B). The ERb-EGFR-caveolin 1 interaction was further investigated by confocal imaging in mock-transfected and in ERb over-expressing REN cells (Fig. 4C). In mock-transfected cells, there was little co-localization of ERb with either EGFR or caveolin 1 within the cytoplasm or at the cell membrane; EGF treatment resulted in a co-localization of ERb with EGFR and caveolin 1 at discrete sites largely located within the cytoplasm of treated cells. In ERb over-expressing REN cells ERb was associated at high abundance with EGFR and caveolin 1 at discrete sites within the cytoplasm, proximal to the cell membrane, supporting co-immunoprecipitation data. The co-localization of the proteins occurred independently of EGF treatment. The redistribution of EGFR to discrete sites was specific to over- expression of ERb and was not observed when ERa was over- expressed in these cells (Fig. 4D). ERb expression influences response of MMe cells to Gefitinib Gefitinib is an EGFR tyrosine kinase inhibitor that acts by binding to the adenosine triphosphate (ATP)-binding site of the enzyme, employed in the treatment of certain types of carcinomas. However, lack of correlation between EGFR expression and response to its tyrosine-kinase (TK) inhibitor Gefitinib has been reported in different malignancies [28,29]. Mutations in EGFR- TK domain have been associated with response in patients with metastatic NSCLC [30]. The prevalence of such mutations in mesothelioma is presently unknown but it seems that they are very rare in mesothelioma [31]. Here we tested if ERb expression could influence response to Gefitinib of MMe cells. The growth- inhibitory effects of 5 mM Gefitinib were evaluated on mock, ERb over-expressing or ERb silenced REN cells and in mock and ERb expressing MSTO-211H cells (Fig. 5A). REN cells were weakly sensitive to Gefitinib, and over-expression of ERb did not significantly affect the sensitivity of these cells. The silencing of ERb expression rendered the cells more sensitive to EGFR antagonism, suggesting that the loss of ERb expression resulted in PLoS ONE | www.plosone.org 4 November 2010 | Volume 5 | Issue 11 | e14110