Puromycin

To produce shRNA lentiviruses, 2 × 10⁶ human embryonic kidney 293T cells (ATCC #CRL-3216) were plated into a 10-cm² plate and transfected 16 hours later with 8 μg of pLKO-shRNAs (Addgene, #10879 for CTR; Sigma-Aldrich, #TRCN0000033697 for RING1B; and Sigma-Aldrich, #TRCN0000014881 for FOXA1), 2 μg of pCMV-VSV-G, and 6 μg of pCMV-dR8.91 plasmids using calcium phosphate. Seventy-two hours after transfection, the viral supernatant was collected, passed through a 0.45 μM polyethersulfone filter, and used to transduce MDA-MB-231 and T47D cells. Specifically, 3 × 10⁵ cells were plated into a six-well plate followed by the addition of viral media with polybrene (8 μg/ml; Millipore-Sigma, #TR-1003-G). Cells were centrifuged for 1 hour at 1000g at 32°C and then incubated overnight with fresh viral media. Viruses were removed and complete culture medium was added for cell recovery. Cells were selected 24 hours after recovery with puromycin (2 μg/ml; Biogems, #5855822) and were maintained in selection. All experiments were performed within 2 weeks after transduction. The shRING1B oligos were cloned into the pLKO-tet-on plasmid, and LT3GEPIR-shRenilla luciferase was used as the doxycycline-inducible control. Lentiviruses were produced from the two plasmids as described above. Cells were treated with doxycycline (100 ng/ml) for 2 days before culturing them in HD media for 72 hours.