Plk0.1 puro

The lentivirus backbone vector pLK0.1-puro, engineered to express Hdac3 (TRCN0000039389), Gata6 shRNA (short hairpin RNA) (TRCN000085589) or non-silencing shRNA control was purchased from Sigma-Aldrich and GE Dharmacon (Lafayette, CO). The lentivirus backbone vector PGIPZ expressing p300 shRNA (V2LMN_102133 and V2LMN_90586) or non-silencing control shRNA was obtained from GE Dharmacon. Mouse Gata6 cDNA was cloned into the lentivirus backbone vector pRRL.hCMV.Sin.IRES.GFP^{63 (link)}. Infectious lentivirus was created by co-transfection of lentivirus vectors expressing Hdac3, Gata6 and p300 shRNAs and Gata6 or control vectors with pCMVΔR8.91 and pMD.G into human 293 T cells. The infection mixture was added dropwise to 293 T cells plated on 100-mm culture dishes and incubated at 37 °C overnight. Virus was harvested 48 h or 72 h after transfection, concentrated with PEG-it virus precipitation solution (System Biosciences, Mountain View, CA) and titered with HIV p24 ELISA (Cell Biolabs, San Diego, CA).

MISSION® shRNA Lentiviral Transduction Particles (Sigma Aldrich, St. Louis, MO, USA, SHCLNV-NM_008685) were used to knockdown the Nfe2 gene expression in the 4T1.3 and TS/A.3 clones. shRNAs were generated against the mouse Nfe2 mRNA (TRC Number: TRCN0000081983 and TRCN0000424174) and were packaged into the lentiviral particles. shRNAs targeting scrambled RNAs (Nontarget; SHC002) were prepared with the use of pLK01-puro (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer’s instructions. Stably gene knocked-down clones were selected in the presence of puromycin (3.5 µg/mL for 4T1.3 cell line and 6 µg/mL for TS/A.3 cell line).