Sihif2 α

About 1x10⁶ cells were washed and resuspended in 100μl Amaxa nucleofection buffer (Lonza). 100nM of the following siRNAs were then added to the mixtures: siARG2 (Ambion, #s1571), siARG1 (Ambion, #s1568), siHIF1−α (Ambion, #s6539), siHIF2−α (Ambion, #s4700), siARNT (Ambion, #s1613), and control siRNA (Ambion, silencer select control siRNA #1). The mixtures were electroporated using the preset K562 ATCC program on the Amaxa nucleofection device (Lonza). Cells were resuspended in 5ml of RPMI media for recovery before further treatments.

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.