Hif 2α

Freshly isolated CB-MNCs were transfected with dssiRNA HIF-1α and HIF-2α (Qiagen, Venlo, Netherlands) using ‘Human monocyte nucleofactor kit' (Lonza, VPA-1007). Briefly, for each condition an equal amount of CB-MNCs was centrifuged, and 100 μl nucleofactor was added to the cell pellet. From both dssiRNA HIF-1α and HIF- 2α, 1 μg was added to the cell suspension, and transferred to the cuvette and placed in the electroporation system (Amaxa, Lonza Verviers) according to the manufacturer. Transfected CB-MNCs were resuspended in complete EGM medium, transferred to 0.1% gelatin coated wells, and further cultured under 1% O₂ according the CB-ECFC culture protocol. Mock transfected cells (only electroporation step, no dssiRNA transfection) served as a control in 20 and 1% O₂. Further details are given in Nauta et al. (30 (link)).

hESCs maintained on Matrigel at 5% oxygen were passaged and incubated overnight. For each transfection, 50 nM siRNA (CTBP1/2 [Ambion]; CTBP1 [Ambion]; CTBP2 [Ambion]; HIF-2α [QIAGEN]), along with 12 μL INTERFERin for CTBP siRNAs (Polyplus) or HiPerFect for HIF-2α siRNA (QIAGEN) transfection reagent were mixed in 200 μL of KnockOut DMEM (Invitrogen) and added in a drop-wise manner to 1 well of a 6-well plate. Cells were harvested 48 h post-transfection and RNA or protein extracted. Allstars control siRNA (QIAGEN) that has no homology to any known mammalian gene was used as a negative control for each transfection.
For double knockdowns (CTBP1+2 siRNA), 50 nM of each siRNA and 12 μL InterferIN transfection reagent were added to 200 μL of KnockOut DMEM. Twice the volume of Allstars negative control siRNA was added to the controls.

U2OS cells were transfected with control siRNA (10 nM; 5′- UUCUCCGAACGUGUCACGU -3′) or siRNA against Arnt (5′-GGAACAAGAUGACAGCCUATT -3′), Miz-1 (5′- GCCUCAUCAGCCUGCUGAATT -3′), Hif1α (5′- GAAGAACUAUGAACAUAAATT -3′) and Hif2α (5′- CGGAUAGACUUAUUGCCAATT -3′) (Qiagen) using the HiPerFect transfection reagent (Qiagen) according to the manufacturer’s instructions. The cells were cultured for 48 hours and then placed at 21% or 1% O₂ for 6 hours before harvested.