Flag pcdna3

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).

To assess relevant amino acid residues in regulating the nuclear translocation activity of MCL1, we used PCR-based cloning to generate each of those deletions and cloning into pcDNA 3.1 vector. Flag-fused MCL1 was cloned into Flag-pcDNA 3.1 (Addgene #52535). The resulting plasmids were sequenced to ensure that they encoded the appropriate constructs. Transient plasmid transfection into the indicated cell lines was performed using Lipofectamine 2000 (Life Technologies, Carlsbad, CA) according to the instructions of the manufacturer. Briefly, 2 × 10⁵ cells/well in six-well plates or 1 × 105 cells/well in six-well plates on poly-l-lysine-coated glass coverslips were transiently transfected with 0.5 μg of WT MCL1, various MCL1-expressing constructs, or empty pcDNA3.1 vector (Invitrogen, Carlsbad, CA). The medium was changed after 24 h, and then cells were incubated for 48 h prior to verifying transgene expression by Western blot analysis. Flag-tagged MCL1 proteins were purified by using M2 affinity agarose gel (Sigma, St Louis, MO) according to the protocols from the manufacturer.