The CK6 expression was found to be concomitant with the proliferation rate

2 / 15 Lasting Effect of HIF-1 on Malignant Progression Lentiviral transduction To establish tetracycline-regulated stable cell lines, we PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19775295 used the ViraPower HiPerform T-REx Gateway Vector kit following the manufacturer’s instruction. Tetracycline regulation in the T-REx system is based on the binding of tetracycline to the tetracycline repressor and derepression of the promoter controlling expression of either HIF1 or HIF2. Cells were infected at 5 multiplicity of infections with the lentivirus expressing tetracycline repressor and selected with geneticin at 500 g/ml for U-2 OS, 200 g/ml for U-87 MG, and 400 g/ml for U-118 MG. These cells were then infected at 2 MOIs with a lentivirus derived from any of the pLenti6.3/TO/V5-DEST constructs and selected with blasticidin at 5 g/ ml for U-2 OS and U-118 MG, and 2 g/ml for U-87 MG. Selected cells were pooled and used for further studies. Cell culture and intermittent induction with tetracycline U-2 OS, U-87 MG, and U118MG were purchased from the American Type Culture Collection. U-2 OS and U-118 MG cells were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum and penicillin/streptomycin. U-87 MG cells were maintained in minimum essential medium with supplements as above. Cell culture conditions were maintained at 37C and 5% CO2. For intermittent induction of the gene of interest, tetracycline was administered weekly at 1 g/ml on day 1 and removed on day 4 for a total of 8 weeks. Western blot Cell extract was prepared in a lysis buffer which contains 20 mM HEPES, pH 7.9, 0.42 M NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol freshly supplemented with 0.5 mM DTT and protease inhibitor cocktail. Protein concentrations were determined by using Pierce BCA protein assay kit. Antibodies used for Western blotting were mouse anti-human HIF-1, rabbit anti-V5 antibody, and anti–tubulin. Signals were developed using Super Signal West Pico chemiluminescent substrate. Gene expression, cell proliferation, and anchorage-independent growth For reporter assays, 293T cells were seeded in 24-well plates and transiently cotransfected with 400 ng MEK 162 pEpoE-luc and 200 ng pLenti-HIF1 or pLenti-HIF2, as well as 50 ng pCMV-EGFP for normalization. pLenti-LacZ was used as a control. Twenty-four hours after transfection, cells were lysed and assayed for reporter activity in the Bright-Glo Luciferase assay system according to the manufacturer’s instructions. Reagents for TaqMan gene expression were purchased from Invitrogen and real-time PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19778700 PCR reactions were performed according to the manufacturer’s instructions. Assays for cell proliferation and anchorage-independent growth were described previously. Tumor transplantation All animal studies were performed according to the protocol approved by the University of Utah Institutional Animal Care and Use Committee. There were six mice in each group or otherwise indicated. For subcutaneous injections, treated U-2 OS cells were suspended in 100 l of phosphate-balanced saline per injection and grafted into the flanks of 68-week-old non-obese diabetic/severe-combined immunodeficient IL-2Rg-null 3 / 15 Lasting Effect of HIF-1 on Malignant Progression ) male mice. Mice were sacrificed and tumor was extracted when the diameter reached 1 cm. For intracranial implantation, treated U-87 MG or U-118 MG cells in a total volume of 5 l were mixed with BD Matrigel basement membrane matrix. NSG mice were anesthetized with isoflurane, and injections were posi