Non targeting sirna 2

HeLa cells were grown to confluence and re-seeded at 150 k/well (6 well plate, Greiner). Next day, cells were siRNA transfected as specified by the manufacturer using 5 μl/well RNAIMAX (Invitrogen, 13778–075) and a final siRNA concentration of 50 nM. Medium was replaced the next day and cells were grown overnight in normoxia or hypoxia and harvested 48 h after transfection. For harvesting, cells were washed twice with cold PBS on ice, scraped in cold PBS supplemented with protease inhibitors (Roche), and pelleted by centrifugation (2600×g, 2 min at 4°C). Protein samples were lyzed in 60 μl of lysisbuffer (0.05 M sodium phosphate pH7.3, 0.3M NaCl, 0.1% NP40, 10% Glycerol). Cell pellets for RNA isolation were frozen in liquid nitrogen and stored in −80°C. siRNAs used in this study: hHIF1α (L-004018–00–0005, Thermo Scientific), non-targeting siRNA #2 (D-001210–02, Thermo Scientific), hARNT siRNA (L-007207–00–0005), hE2F7 (HSS135118, HSS135119, HSS175354, Invitrogen), hE2F8 (HHS128758, HSS128759, HSS128760, Invitrogen), Negative control medium (Invitrogen, 12935–300). The negative control siRNAs from Thermo and Invitrogen were mixed and used at a 1:1 ratio. For double transfection, half of the amounts applied for the single siRNA transfections were used, ensuring a final concentration of 50 nM. For siRNA transfections in U2OS cells were seeded at a density of 100 k/well.