Lentiviral Knockdown of HIF-1α, HIF-2α, TAZ, and SIAH1

Vectors encoding shRNA targeting HIF-1α (sh1α1) and HIF-2α were previously described [21 (link)]. The shRNA vectors encoding NTC and sh1α2 were purchased from Sigma. pLKO.1-puro lentiviral vectors encoding shRNA targeting TAZ and SIAH1 were purchased from Sigma-Aldrich: shT1 (Clone ID: NM_015472.3-706s1c1); shT2 (Clone ID: NM_015472.3-1657s21c1); shSI1-1 (Clone ID: NM_003031.3-563s21c1); shSI1-2 (Clone ID: NM_003031.3-826s21c1); shLA2-1 (Clone ID: NM_014572.x-3750s1c1) and shLA2-2 (Clone ID: NM_014572.x-2162s1c1). Lentiviral vectors were co-transfected with plasmid pCMV-dR8.91 and a plasmid encoding vesicular stomatitis virus G protein into 293T cells using Lipofectamine 2000 (Invitrogen). Medium containing viral particles was collected 48 h after transfection and passed through a 0.45-μM filter. MDA-MB-231, MDA-MB-435 and MCF-7 cells were transduced with viral supernatant supplemented with 8 μg/mL of Polybrene (Sigma-Aldrich). After 24 h, cells were selected in medium containing 0.6 μg/mL of puromycin (Sigma-Aldrich).