viability and cell cycle regulation. PHD3-specific inhibition could be a feasible mean to block the hypoxia-induced cell survival at least in squamous cell carcinomas. Materials and Methods Patient samples Tumor samples were collected, after informed written consent, from surgically removed oral and oropharyngeal HNSCC in Turku University Hospital, Finland. Normal tissue samples used as a control were collected from patients undergoing uvulopalato- pharyngoplasty. Samples were from both genders and the age of the patients ranged from 29 to 87 years. All procedures in this study were approved by the National Authority for Medicolegal Affairs and by the ethics committee of Turku University Hospital. All data were analyzed anonymously. The patient material has been described in detail elsewhere [52,53]. For database studies OncomineTM was used [36]. Cell culture and transfections HeLa cells were obtained from the American Type Culture Collection, Rockville, MD, USA. The cancer patient-derived HNSCC cell lines UT-SCC2, UT-SCC7 and UT-SCC9 used in this study have been at the University of Turku and described previously [35]. Cells were cultured in DMEM, supplemented with 10% FCS, penicillin-streptomycin and L-glutamine. The SCC cells were also supplemented with nonessential amino acids (Sigma-Aldrich). Cells were cultured in humidified air condition- ing 5% CO2 at 37uC. For hypoxia treatments air was replaced by nitrogen to reach 1% oxygen in a hypoxia workstation (Invivo2). For siRNA experiments, double stranded siRNA oligonucleo- tides were used at 200 nM final concentration. Transfections were performed with Oligofectamine (Invitrogen) following the manu- facturer’s protocol. The siRNAs (MWG Biotechnology) used were: siPHD2 59-GACGAAAGCCAUGGUUGCUUG (dTdT)-39; siP- HD3 59-GUCUAAGGCAAUGGUGGCUUG(dTdT)-39; siPH- D3 off-target 59-GUC UAAGGCAUAGGUGGCUUG(dTdT)-39 and non-target (scr) siRNA 59-CCUACAUCCCGAUCG AU- GAUG(dTdT)-39. For some of the cell cycle experiments the cells were synchronized using aphidicolin at 1 mg/ml. 18 to 22 hours after incubation the cell cycle was released by washing the cells with PBS three times and adding fresh media to the cells. mRNA, western blot analysis and antibodies RNA was extracted using RNeasy mini kit (Qiagen). Q-RT- PCR (Taqman) cDNA was prepared with M-MMLV RT (H2) (Promega) and NTP-mix (Fermentas) at 0,5 mM and analyzed according to manufacturer’s instructions (Applied Biosystems). Taqman primers (Eurogentec) used were: PHD3 fwd 59 – CG- AAGTGCAGCCCTCTTACG – 39; PHD3 rev 59 – TTT- TGGCTTCTGCCCTTTCTT – 39 and PHD3 probe (Euro- gentec) 59 – AACCAGATATGCTATGACTGTCTGGTACTT- TGATGCT – 39. The expressions were normalized against EF- 1a. For protein analysis cells were harvested in SDS-Triton lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% Triton X- 100, 5% glycerol, 1% SDS, 1 mM Na3VO4, 1 mM phenyl- methylsulphonyl fluoride and 10 mM NaF,). Protein concentra- tions were measured with Bio-Rad DC protein assay before addition of 66SDS buffer. Proteins were detected by Western blotting and enhanced chemiluminescence with specific antibod- ies: active caspase 3 (G748A, Promega, 1:500), cyclin B1 (sc-752, Santa Cruz Biotechnology, 1:2000), cyclin D1 (sc-246, Santa Cruz Hypoxic Cell Cycle Arrest by PHD3 Inhibition Biotechnology, 1:2000), HIF-1alpha (610959, BD Transduction Laboratories, 1:3000), PHD2 (NB 100–137, Novus Biologicals, 1:3000), PHD3 (NB 100–139, Novus Biologicals, 1:2000) and Ac- 74 b-actin antibody (Sigma-Aldrich, 1:4000). Secondary anti- mouse-HRP and anti-rabbit-HRP (DAKO) antibodies were used at 1:10 000. Cell proliferation, imaging and immunocytochemistry For BrdU proliferation assay the cells were transfected with corresponding siRNA and plated on 96-well plates (8 wells per siRNA). Plates were incubated in normoxia or in hypoxia for 48 hours. BrdU was added and incubated for 4 hours. The cell proliferation was measured with colorimetric ELISA, BrdU assay (Roche Diagnostics). BrdU assay was performed according to manufacturer’s instructions. For cell counting the cell nuclei cells were fixed with PTEMF fixing solution (100 mM PIPES pH 6.8, 10 mM EGTA, 1 mM MgCl2, 0,2% Triton X-100 and 4% formaldehyde) and stained with the nuclear stain Hoechst 33342 (Invitrogen). Optical fields of cells were imaged with Zeiss Lumar V12 fluorescence stereo microscope (Carl Zeiss) and the number of nuclei per optical field was calculated using ImageJ software (NIH, USA) nucleus calculator option. Experiments were done as parallel treatments and each experiment was repeated at least three times. For detection of active caspase 3 the cells were stained with caspase 3 antibody described earlier. Flow cytometry Cell cycle and apoptosis were detected with flow cytometer (BD FACSCalibur, BD Biosciences). The samples were collected by trypzination, washed and fixed with 70% ethanol in 220uC for 24 h. The fixing solution was removed by centrifugation and nuclei were washed with PBS. Staining was done in RT with propidium iodide (PI) (10 mg/ml) in PBS with 0,1% Tween. The cell cycle histograms were analyzed using BD CellQuest Pro software (BD Biosciences). Statistics For statistical analysis Spearman correlation and two-tailed Student’s t-test were used. Supporting Information Figure S1 Hypoxia induces the expression of PHD2 but not PHD1 in UT-SCC. Five different primary human head and neck squamous cell carcinoma-derived cell lines (SCC) were cultured in normoxia or in hypoxia for 6 and 48 hours. The cells were exposed to normoxia or hypoxia for 6 and 48 hours. The PHD mRNA expression was detected and quantified by Q-RT- PCR. (A) PHD1 mRNA levels reduced during prolonged hypoxic exposure whereas (B) the PHD2 levels were induced in short hypoxia. (C) PHD3 mRNA levels under siPHD3 exposure in normoxia (left-hand panel) and hypoxia (right-hand panel). Please note the different scale for normoxia and hypoxia. (TIF) Figure S2 Expression of transfected EGFP, PHD3-EGFP and PHD3R206K-EGFP (Fig. 3) studied by confocal microscopy. (TIF) Figure S3 Hypoxia-activated apoptosis is not enhanced by PHD3 inhibition. The apoptosis rate was determined by flow cytometer at the indicated time points. The hypoxic exposure PLoS ONE | www.plosone.org 10 November 2011 | Volume 6 | Issue 11 | e27112