cultured in RPMI medium supplemented with 10% foetal bovine serum (FBS) at 37uC in a 5% CO2-humidified atmosphere. For experimental purposes, the cells were maintained in the same culture medium but lacking phenol red and containing charcoal- stripped FBS. Mycoplasma infection was excluded by the use of the Mycoplasma PlusTM PCR Primer Set kit from Stratagene (La Jolla, CA). Cells grown to 80% confluence in tissue culture dishes were transiently transfected with the pCXN2 plasmid expressing human wild type ERb (Addgene, Cambridge, MA) using LipofectAMINE reagent as described by the manufacturer. Gene silencing was achieved using an ERb-specific shRNA lentiviral plasmid (pLKO.1-puro) by Sigma (St Louis, MO). Cell lysis, immunoprecipitation and immunoblot Cells were extracted with NP-40 lysis buffer (1% NP-40, 150 mM NaCl, 50 mM Tris-HCl pH 8, 5 mM EDTA, 10 mM NaF, 10 mM Na4P2O7, 0.4 mM Na3VO4, 10 mg/ml leupeptin, 4 mg/ ml pepstatin and 0.1 Unit/ml aprotinin). Cell lysates were centrifuged at 13.000 x g for 10 minutes and the supernatants were collected and assayed for protein concentration using the Bradford protein assay reagent (Bio-Rad). Proteins were separated by SDS-PAGE under reducing conditions. For co-immunoprecip- itation experiments, cells were incubated with antibodies specific for EGFR for 1 hour at 4uC, then lysed and a volume equivalent to 2 mg of extracted protein for each treatment was incubated in the presence of 50 ml protein A-Sepharose beads. Following SDS- PAGE, proteins were transferred to nitrocellulose, reacted with the specific antibodies indicated and then detected with horseradish peroxidase-conjugated secondary antibodies and the chemiolumi- nescent ECL reagent. Densitometric analysis was performed using the GS 250 Molecular Imager (Bio-Rad). For Cyclin D1 expression, cells were extracted in RIPA Buffer (1% Triton X-100, 0.1% SDS, 1% Na-deoxycholate, 150 mM NaCl, 50 mM Tris-HCl pH 7, 0.4 mM Na3VO4, 10 mg/ml leupeptin, 4 mg/ml pepstatin and 0.1 Unit/ml aprotinin) and analyzed as indicated above. Cell proliferation as determined by direct counting REN or MSTO-211H cells were seeded at a density of 16104 cells/well into six-well plates in growth medium supple- mented with FBS and incubated overnight at 37uC in a humidified environment containing 5% CO2 to allow the cells to become adherent. Cells were alternatively transiently transfected with the pCXN2 ERb plasmid or ERb shRNA, using the LipofectAMINE reagent as described by the manufacturer. After 24 hours cells were grown for a further 24–48 hours as indicated in complete medium or treated with 5 ng/ml of human recombinant EGF in 2% charcoal-stripped FCS growth medium. Cells were then trypsinized and stained with Trypan blue. The number of viable cells was counted in a Burker chamber within 5 min of staining. The same protocol was used to evaluate drugs sensitivity. Assay for Anchorage-Independent Cell Growth Anchorage-independent growth was determined using a modifica- tion of previously described methods [40]. Briefly, a base layer of 0.6% agar in complete medium was plated in six-well plates and allowed to solidify (0.5 mg/ml Puromycin was added to wells containing shERb transfected cells, while 250 mg/ml G418 was added to ERb transfected cells). Next, wells were overlaid with 56103 cells per well in a 0.3% agar. A growth control well was also included with 56103 cells in medium alone (no agar) for each cell line. The plates were incubated at 37uC, 5% CO2 for 15 days and checked every 2 days for colony formation. At day 7, individual colonies (defined as clusters of 15 or more cells) were counted in 10 random fields. ERb Function in Mesothelioma Confocal microscopy analysis Immunofluorescence was performed using standard techniques. Briefly, cells were plated (16105 cells) on glass cover slips and allowed to adhere in a humidified atmosphere with 5% CO2 at 37uC. Wild type or transfected cells were then stimulated with EGF (5 ng/ml for 5 minutes) and subsequently fixed with 4% paraformaldehyde in PBS. Cells were permeabilized 5 minutes at RT with 0.2% Triton-X in PBS. After fixation, the cells were rinsed in PBS and incubated in a blocking solution containing 1% Gelatin and 4% bovine serum albumin (BSA) in PBS for 1 hour. Primary antibodies diluted in 2% BSA in PBS, were added in combination to the fixed cells and incubated at room temperature for 2 h. After washing in 2% BSA in PBS, the immunoreactivity was revealed using Alexa Fluor 488 goat anti-mouse IgG or tetramethylrhodamine isothiocynate (TRITC) goat anti-rabbit secondary antibodies (Invitrogen, Paisely, UK) in 2% BSA in PBS (1:200) used separately to stain the cells for 30 min at room temperature. Negative controls were performed by substituting the primary antibodies with the 2% BSA in PBS buffer. The immuno- stained cells were rinsed with PBS and mounted in Vectashield mountant (Vector Laboratories, Burlingame, CA) containing 49-6- Diamidino-2-phenylindole (DAPI). Confocal imaging was per- formed using a laser scanning LSM 510 confocal microscope (Carl Zeiss, Welwyn Garden City, UK). Alexa Fluor 488, TRITC and DAPI fluorophores were excited individually at 488 nm, 543 nm and 364 nm respectively. Single focal plain scans of 0.8 mM depth were performed at the mid diction of the cell monolayer using the 63x1.4 oil immersion objective. Internalization assay by flow cytometry analysis Cell surface EGFR expression was evaluated by flow cytometry performed as described. Cells were grown in a Petri dish until confluent, washed with PBS, detached using 0.1% trypsin–EDTA and re-suspended in PBS with 1 mM CaCl2 and 1 mM MgCl2 supplemented with 4% FBS. After the indicated treatments, cells were incubated for 30 minutes at 4uC with the monoclonal anti- EGFR antibody or non-specific IgG as control, washed twice with PBS and further incubated with fluorescein isothiocynate (FITC)- conjugated goat anti-mouse secondary antibody (1:200) for 30 minutes. After washing twice, cells were fixed with 3% paraformaldehyde in PBS at room temperature for 15 minutes and washed twice in PBS. FITC fluorescent emission was detected over the range 515–555 nm using the FL-1 detector of a FACScan flow cytometer (Becton Dickinson, Franklin Lakes, NJ) equipped with 15 mW argon–ion laser for excitation. Debris was gated out by establishing a region around the population of interest on the Forward Scatter versus Side Scatter dot plot. For each sample, 10.000 events in the region of interest were recorded at a flow rate of 200–300 cells/s. Data were processed with analysis software LYSYS II (Becton Dickinson) and are expressed as median value of EGFR expressing cells of the fluorescence histograms normalized to the corresponding negative control obtained by omitting the primary antibody. Statistical analysis Statistical differences between treatment groups were measured using the one-tailed Student’s test. Author Contributions Conceived and designed the experiments: GP WT L. Moro. Performed the experiments: GP WT PB AGM. Analyzed the data: GP WT REF BH L. Mutti, L. Moro. Contributed reagents/materials/analysis tools: REF BH L. Mutti, L. Moro. Wrote the paper: BH L. Mutti, L. Moro. PLoS ONE | www.plosone.org 9 November 2010 | Volume 5 | Issue 11 | e14110