tions of expression vectors or RNA interference were performed as previously reported [44], using Lipofectamine (Invitrogen). SureSilencing shRNA plasmids against HSP90a and b isoforms containing the hygromycin resistance gene were obtained from SABiosciences (Frederick, MD). Polyclonal population of stably transfected cells were used. Viruses were generated as previously described [45]. In short, the Phoenix amphotropic packaging line was transfected with the pBabe-based retroviral expression vectors carrying wild type (Addgene plasmid 19365) or hydroxylation- resistant (P402A/P564A) form (Addgene plasmid 19005) of HA- tagged HIF-1a. Transfected cells were incubated for 48 h at 37uC for virus production. The virus-containing medium was collected, filtered and used to infect the target cells. Stable clones or mixed populations were cultured in the presence of puromycin (1 mg/ml). Reagents Cyclohexamide (CHX), Z-leu-leu-leu-CHO (MG132), Cobalt Chloride (CoCl2), Desferrioxamine (DFO), 17-Allylamino-17- demethoxy-geldanamycin (17-AAG), insulin, Epidermal Growth Factor (EGF) were purchased from Sigma-Aldrich (St. Louis, MI, USA). Isolation of nuclear/cytoplasmic fractions Nuclear and cytoplasmic fractions were prepared as follows: 1– 26106 cells were resuspended in a hypotonic lysis buffer (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA) containing protease inhibitors (Boehringer). After resuspension, NP-40 was added to a final concentration of 0.6% and the nuclei were isolated by centrifugation at 10,000 r.p.m. for 30 s at 4uC. After removing the supernatant (i.e. the cytoplasmic extract), the nuclei were re-suspended in a nuclear extract buffer (20 mM HEPES pH 7.9, 25% glycerol, 0.4 M NaCl, 0.1 mM EDTA, 0.1 mM EGTA), rocked for 15 min at 4uC and then recovered by centrifugation at 140,00 r.p.m. for 5 min at 4uC. Immunoprecipitation and Western blot analysis For immunoprecipitation assays and Western blot analysis, the cells were lysed in 0.3% CHAPS buffer (40 mM HEPES [pH 7.5], 120 mM NaCl, 1 mM EDTA, 10 mM pyrophosphate, 10 mM glycerophosphate, 50 mM NaF, 1.5 mM Na3VO4, 0.3% CHAPS, and one tablet EDTA-free protease inhibitors [Roche] per 10 ml). Followed by centrifugation, the supernatant was precleared with protein A/G agarose beads coupled with mouse or rabbit IgG (Pierce, Thermo Fisher Scientific, Rockford, IL) for .2 h and then was exposed to 1 mg of the antibody (Santa Cruz Biotechnology, Santa Cruz, CA) or mouse or rabbit IgG, as control, was added to each of the cellular lysates and incubated overnight at 4uC followed by incubation with protein A/G-agarose beads (Amer- sham Biosciences Europe, Milan, Italy) for 2 h at 4uC. Immunoprecipitates were washed four times in the lysis buffer before Western blotting analysis. For some immunoprecipitation experiments we used ExactaCruzTM reagents (Santa Cruz Biotecnology) to detect the bcl-2 protein without detection of the light chain of the immunoprecipitation antibody. Immunoprecip- itation were also performed using multiple antibodies recognizing different epitopes on the bcl-2 (Santa Cruz Biotecnology) and HIF- 1a (Santa Cruz Biotecnology; Novus Biologicals, Littleton, CO) protein. Sequential immunoprecipitation experiments were per- formed incubating 2 mg of total cell lysate with antibody as for single immunoprecipitation, after washing the precipitated pro- teins were released with 1% SDS at 37uC for 30 minutes. Then, the eluate was diluted to a final concentration of 0.1% SDS with lysis buffer and immunoprecipitation was repeated with the supernatant with fresh beads and antibody. HIF-1a Stabilization by Bcl-2 For Western blot analysis, antibodies directed to HIF-1a, HIF- 1b, HSP90 (BD Pharmingen), HA epitope, ubiquitin (Santa Cruz Biotecnology), bcl-2 (Dako, Milan, Italy), b-tubulin (Thermo Scientific), HSP90a, HSP90b (Abcam, Cambridge, UK), PHD2 (Novus Biologicals), Lamin A/C (Cell Signaling, Danvers, MA), b- actin (Sigma) were used. Pulse and pulse-chase assays In the pulse assay, cells were incubated with methionine/ cysteine–free serum-free DMEM (Invitrogen) for 2 h. [35S]- labeled methionine-cysteine (88 mCi/ml, EasyTagTM EX- PRESS35S Protein Labeling Mix, PerkinElmer, Waltham, MA) was added to the medium and cells were collected after 15 and 45 min. In the pulse-chase assay, after 45 min pulse with [35S]-labeled methionine-cysteine, cells were washed three times with PBS, chased with DMEM containing 10% FBS and 2.5 mg/mL cold L-methionine and harvested after 15, 30, 45 and 60 min. Total protein lysates from pulse and pulse-chase assays were immunoprecipitated by HIF-1a antibody. Radio- labeled HIF-1a protein and the input cell lysates were subjected to SDS-PAGE. Gels were dried, exposed in phosphorImager cassette for 1–3 days and imaged using Personal Molecular Imager FX and Quantity OneH software (Biorad Laboratories, Hercules, CA). ELISA The supernatants were harvested and assayed for VEGF content by ELISA kit according to the manufacturer’s instructions (R&D Systems, Minneapolis, MN). VEGF levels were normalized to the number of adherent cells. Reporter gene assay The cells were seeded in 24-well plates and were transfected with a total of 1 mg of DNA/well using Lipofectamine reagent. The evaluation of HIF-1 transcriptional activity was performed as previously described [12] transfecting cells with a vector expressing luciferase under the control of 4X Hypoxia Responsive Element (HRE) and another one expressing b-galactosidase under the control of CMV promoter. The relative luciferase activity was calculated by luciferase/b- galactosidase ratios for each sample. Confocal analysis After 24 h hypoxic conditions exposure, cells were fixed in 100% methyl alcohol for 10 min at 220uC and then incubated with primary antibodies. The cells were incubated with TRITC conjugated Goat anti-Rabbit and/or FITC conjugated Goat anti- Mouse (Jackson Lab, West Grove, PA). Nuclei were visualized using TO-PRO3H (Invitrogen). The images were scanned under a 640 oil immersion objective and to avoid bleed-through effects, each dye was scanned independently by a Leica confocal microscope (laser-scanning TCS SP2) equipped with Ar/ArKr and HeNe lasers. The images were acquired and electronically merged utilizing the Leica confocal software (Leica Microsystems Heidelberg GmbH, Mannheim, Germany). Figures were pro- cessed using Adobe PhotoShop software. Densitometric analysis Developed films were acquired using GS-700 Imaging Densi- tometer (Biorad) and processed with Corel Photo Paint 7.0 to adjust image brightness and contrast. Densitometric evaluation was performed using Molecular Analyst Software (Biorad) and normalized with relative controls depending on the analysis performed. PLoS ONE | www.plosone.org 11 July 2010 | Volume 5 | Issue 7 | e11772