Lentivirus transduction.

To develop cell lines with nuclear fluorescence for live imaging, lentiviral stocks were developed by the University of Michigan Vector Core using an mKate2 2X nuclear localization fusion construct. Tumorigenic MDA-MB-231, NCI-H460, and LNCaP cells were seeded at 3 × 10⁵ cells/well on 6-well plates (Corning) overnight. The following day, cells were washed in PBS and cell media was replenished with 1.35 mL fresh media, 150 μL 10× lentiviral stock supernatant (MOI of approximately 6), and 4 μg/mL Polybrene (Sigma-Aldrich). Plates were incubated at 37°C with 5% CO₂ for 24 hours. Subsequently, mKAte2-transduced cells were washed in PBS and expanded in culture until optimal confluency was achieved.

To isolate individual clones of transduced cells, fluorescent MDA-MB-231, NCI-H460 and LNCaP cells were singly sorted into individual wells of a 96 well plate (Corning) at the University of Michigan Flow Cytometry Core using a FACS Synergy Head #1 cell sorter (Sony Biotechnology). The viability marker Zombie Violet was used to exclude dead cells. The cells sorted were negative for Zombie Violet (450/50 (405)) and in the top 5% of mKate fluorescence intensity (615/30(561)).

To monitor tumor growth by bioluminescence imaging in vivo, MDA-MB-231 breast cancer cells were transduced with a luciferase lentivirus reporter regulated by the CMV promoter (purchased from the University of Michigan Vector Core). Transduction was carried out by centrifuging 2 × 10⁶ cells in 1 mL media (1,000g for 2 hours) with addition of 8 μg/mg Polybrene and 1 mL 10× luciferase virus (MOI of approximately 6). Culture media was replaced with fresh, warm media after 18 hours, and luciferase expression was analyzed 5 days later using a Dual-Luciferase Reporter Assay System (Promega) and bioluminescence imaging.