Teto fuw empty vector

Transduction constructs were produced as previously described (12 (link), 60 (link), 63 ). Sections of the coding region for PBRM1-containing BD4 mutations were synthesized (Biomatik) and inserted into the digested TetO-Fuw-PBRM1 WT plasmid (Addgene plasmid #85746) (63 ) using the In-FusionHD cloning kit (Clontech Laboratories, Inc) and confirmed by WideSeq (Purdue University). A TetO-Fuw empty vector (Addgene plasmid #85747) (63 ) was also used. The dual reporter construct pFU-Luc2-eGFP (L2G) (77 (link)) was a gift from Huiping Liu. Lentiviral particles for pLenti CMV rtTA3 Hygro (w785-1) (Addgene plasmid #26730) were a gift from Eric Campeau.

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.