Block it fluorescent control sirna

Knockdown of HIF subunits was achieved by using a reverse siRNA transfection procedure performed in six-well plates. Therefore, for each well to be transfected 9 μl Lipofectamine RNAiMAX (Life technologies, ThermoFisher Scientific) were mixed with 150 μl Opti-MEM (Gibco) and combined with 200 pmol siRNA (BLOCK-iT Fluorescent Control siRNA, #442926, Life technologies; siHIF-1α, #L004018-00, Dharmacon, GE Healthcare, Lafayette, CO, USA; siHIF-2α, #4390825, Ambion, ThermoFisher Scientific) diluted in 150μl Opti-MEM (Gibco). The transfection mixture was incubated at room temperature and cells were harvested in RPMI 1640 supplemented with 10% FBS without antibiotics. In total, 4 × 10⁵ Hep3B cells/well were mixed with the transfection mixture followed by the addition of 1ml antibiotic-free growth medium and incubated overnight. On the next day, the supernatant was replaced by 2 ml fresh antibiotic-free growth medium and cells were exposed to normoxia or hypoxia for 8 h.

Gene silencing was achieved by a reverse transfection procedure using Lipofectamine® RNAiMAX (Life technologies) in accordance with the manufacturer’s guidelines. Therefore, cells were seeded on 24-well plates and mixed with the transfection mixture containing either ARNT siRNA (#s1613, Ambion) or BLOCK-iT™ Fluorescent Control siRNA (#442926, Life technologies). Subsequently cells were incubated overnight and subjected to irradiation. To gain protein lysates for Western blot analysis, the transfection procedure was performed in 6-well plates.