Hif1α small interfering rna sirna

RAGE short hairpin RNA (shRNA), ATG5 shRNA, Beclin1 shRNA, p65 shRNA, and control shRNA were obtained from Sigma, whereas pUNO1-RAGE cDNA was obtained from InvivoGene (San Diego, CA, USA). These shRNAs or cDNA were transfected into cells using the Lipofectamine 2000 reagent (Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. To generate stable shRNA-expressing lines, positive cells were selected with 1–2 μg/ml puromycin for two to three weeks. HIF1α- small interfering RNA (siRNA) and control siRNA from Santa Cruz Technology were transfected into cells using X-tremeGENE siRNA reagent (Roche Applied Science, Stockholm, Sweden) according to the manufacturer's instructions.

The human prostate cancer cell lines PC3, DU145, and 22RV1 were purchased from American Type Culture Collection (Manassas, VA, USA). All cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Sigma-Aldrich, St. Louis, MO, USA). All cells were incubated at 37°C in a humidified atmosphere containing 21% O₂ and 5% CO₂. For hypoxic culture, the cells were placed in a hypoxic incubator (1% O₂, 5% CO₂) at 37°C for 6 h. HIF-1α small interfering RNA (siRNA) and negative siRNA were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Propofol was purchased from Sigma-Aldrich.