Small interfering rna oligonucleotides

Small Interfering RNA oligonucleotides were purchased from Eurofins Genomics (Ebersberg, Germany) and used in a final concentration of 27 nM. siRNA transfections in all cell lines were performed using Interferin (Polyplus, Illkirch, France) according to manufacturer’s instructions. Oligonucleotide sequences used for siRNA knockdown are as follows: Control- AACAGUCGCGUUUGCGACU; HIF-1β_#1- GGUCAGCAGUCUUCCAUGA; HIF-1β_#2- GAAAGAAACAUGUGAGUAA; HIF-1α - GCAUAUAUCUAGAAGGUAU; HIF-2α_#1- CAGCAUCUUUGAUAGCAGUTT; HIF-2α _#2- GGCAGAACUUGAAGGGUUA; AHR_#1- UACUUCCACCUCAGUUGGCTT; AHR_#2- GGACAAACUUUCAGUUCUU.
To perform DNA plasmid overexpression in all cell lines, TurboFect (ThermoFisher, Paisley, UK) or GeneJuice (Novagen/ThermoFisher, Paisley, UK) were used according to manufacturer’s instructions. The following expression plasmids were used in this study: GFP-C3 (Clontech/Takara, Montain View, CA, USA); Flag-pcDNA3.1 (a gift from Stephen Smale, Addgene plasmid #20011, Watertown, MD, USA); pEBB-HIF-1β−GFP (kind gift from Colin Duckett, Ann Habour, MI, USA); pCMV5-FLAG TRAF6 (MRC-PPU reagents, Dundee, UK).

Small interfering RNA oligonucleotides were purchased from Eurofins Genomics and were used in a final concentration of 27 nm. siRNA transfections in all the cell lines were performed using Interferin (Polyplus, Illkirch, France) according to manufacturer’s instructions. Oligonucleotide sequences used for siRNA knockdown are: control, 5′-CAGUCGCGUUUGCGACUGGTT-3′; p100/p52, 5′-CUACGAGGGACCAGCCAAG-3′; RelB, 5′-AAUUGGAGAUCAUCGACGAGU-3′; HIF-1α, 5′-GCAUAUAUCUAGAAGGUAUTT-3′; HIF-2α, 5′-CAGCAUCUUUGAUAGCAGUTT-3′.