Plenti cmv rtta3 hygro w785 1

Caki-2 cells expressing GFP and Caki-2 cells re-expressing PBRM1 were generated as previously described (63 ). GFP expression was performed by transducing Caki-2 parental cells with lentiviral particles for the dual reporter construct pFU-Luc2-eGFP (L2G) (77 (link)). GFP-expressing cells were selected using fluorescence-activated cell sorting. GFP⁺ Caki-2 cells were then transduced with lentiviral particles for pLenti CMV rtTA3 Hygro (w785-1) (Addgene plasmid #26730) for tetracycline-inducible expression and selected as described above. Upon selection, cells were transduced with lentiviral particles for TetO-Fuw empty vector (Addgene plasmid #85747). PBRM1 reexpression was performed by transducing Caki-2 parental cells with lentiviral particles for pLenti CMV rtTA3 Hygro (w785-1) (Addgene plasmid #26730) for tetracycline-inducible expression and selected as described above. Upon selection, cells were transduced with lentiviral particles for TetO-Fuw empty vector (Addgene plasmid #85747), TetO-Fuw-PBRM1 WT (Addgene plasmid #85746), or TetO-Fuw-PBRM1-BD4 missense variants. All Caki-2 cells were cultured with 2 μg/ml doxycycline (EMD Chemicals) for at least 72 h before and throughout the experiments to induce protein expression. Cell lines were free of mycoplasma contamination for all experiments.

PBRM1 knockdowns were performed using an empty pLKO.1 vector or pLKO.1 vector containing shRNA to human PBRM1 (TRCN0000015994, ThermoFisher Scientific, Waltham, MA). PBRM1 re-expression was performed by cloning full length PBRM1 from pBabepuroBAF180 (a gift from Ramon Parsons Addgene plasmid # 41078) into tet-inducible conditional lentiviral vector TetO-FUW (a gift from Rudolf Jaenisch Addgene plasmid # 20323), which was used with pLenti CMV rtTA3 Hygro (w785-1) (a gift from Eric Campeau Addgene plasmid # 26730) for tetracycline inducible expression.

All plasmids for the Dual Luciferase Reporter were generated by subcloning gene body (GAPDH) or 5’UTR elements (ER, Myc) into Addgene 45642, pLenti CMV rtTA3 Hygro (w785-1) (Addgene: 26730), pCW57.1-4EBP1_4xAla (Addgene: 38240).