Opti mem media

Packaging plasmids psPAX2 (Plasmid # 12260) and pMD2.G (Plasmid # 12259) were purchased from Addgene. Lentiviral plasmids and packaging plasmids were assembled in Opti-MEM media (Corning) and transfected into HEK293T cells using the TransIT-Lenti Transfection Reagent (Mirus) according to the manufacturer’s instructions. The mass ratio of psPAX2 to pMD2.G used for packaging was 3:1. Media was collected 48 hours after transfection and passed through a 0.45-μm filter and concentrated using Amicon Ultra-15 centrifugal filter (30 kDa, Millipore). Cells were transduced for 16 h followed by 48–72 h of puromycin selection or 6 days of blasticidin selection, depending on the vector. For puromycin, 2 μg/mL was used for HT29 cells and 3 μg/mL was used for DLD1 cells. For blasticidin, 5 μg/mL was used for HT29, COLO205, and DLD1 cells, refreshed every 72 h. We aimed for low MOI infections by empirically determining lentivirus dilution factors to achieve <50% cell survival during antibiotic selection.

In a 24-well plate, 2 × 10⁵ PC3 and C4-2B cancer cells were seeded into each well and allowed to adhere overnight. Lipofectamine 2000 (ThermoFisher; 11668019) was used to deliver 1 µg of plasmids responsive to active (phosphorylated) STAT-1 (pGAS/ISRE Luc) (Signosis; Santa Clara, CA, USA, LR-2016) or STAT-3 (pSTAT-3-Luc) (Signosis; LR-2004) following the manufacturer’s protocols. Transfection media was aspirated, cells were washed with 1 × DPBS, then cells were allowed to recover overnight in complete RPMI media (10% FBS, 1% anti/anti) 37 °C and 5% CO₂. Transfected PC3 cells were serum starved for 5–6 h by replacing complete RPMI media with OptiMEM media (Corning Life Sciences; Corning, NY, USA). Following serum starvation, PC3 cells were treated with targeted Nluc-27.pepL CM for 16 h, while C4-2B were treated for 35 h. Cells were trypsinized then centrifuged at 1500 rpm for 5 min to form pellets. Cells were then lysed with 40 µL 1× passive-lysis buffer, lysates were kept on ice, then 50 µL luciferin substrate were added, and luminescence was measured using a GloMax plate reader (Promega) with 10 s integration time.