Pspax 2 lentiviral packaging plasmid

293T packaging cells were seeded at 1.0 × 10⁶ in 60 mm culture dishes and incubated for 24 hours. PSPAX2 lentiviral packaging plasmid (#12260), pMD2.g vsv-g envelope plasmid (#12259), and scramble shRNA control (#1864) were purchased from Addgene. HDAC2 shRNA were purchased from Sigma (SHCLNG-NM_001527). 2.5 ug PSPAX2, 2.5 ug pMD2.G, 5 ug shRNA, 250 uL serum free DMEM, and 20 uL Fugene 6 (Promega E2691) were added to a 1.7 mL tube and set for 30 minutes at room temperature. The reaction mixture was added to the 60mm dishes and incubated at 37 degrees for 18 hours. Media was replaced with DMEM containing 30% FBS and incubated for another 72 hours. The supernatant was collected and filtered through a 0.45 uM cellulose acetate membrane. To a 100 mm dish, LPS246 were seeded at 1.0 × 10⁶. Thirty minutes prior to infection, polybrene was added to the plate for a final concentration of 8 ug/mL. 1mL of the filtered viral supernatant was added to each plate according to each intended treatment. After 72 hours, the media was replaced and puromycin was added to reach a final concentration of 3.0 ug/mL. Following another 72 hours, the cells were collected for analysis.

HEK293 cells were transfected using FuGENE HD transfection reagent (Promega) with either a wildtype or P2229R mTOR expression vector (ratio of reagent to DNA is 3:1) following the manufacturer’s instruction. Neomycin resistant clones were selected after the cells were cultured with G418 (500 µg/mL) for 3 weeks. The lentiviruses were produced by co-transfection of HEK293FT cells with NFκB2 (wildtype or P882Q mutant) or TLR2 (wildtype or W558L mutant) lentiviral expression vectors, and psPAX2 lentiviral packaging plasmid (Addgene) and pCMV-VSV-G envelope plasmid (Addgene) using Lipofectamine® 2000 (Thermo Fisher Scientific). Antibiotic-free DMEM containing 10% FBS was used as a culturing medium and Opti-MEM I Reduced Serum Medium (Thermo Fisher Scientific) supplemented with 5% FBS and 1 mM Sodium pyruvate was used as a lentivirus packaging medium. Six hours of post-transfection, medium was removed and replaced with DMEM. After 48 h, the supernatants were centrifuged at 300×g for 5 min to remove cell debris and filtered with a 0.45 µm polyethersulfone membrane filter. Ultracentrifugation to concentrate the virus was performed for 2 h at 12,000×g and 4 °C using Beckman SW28 rotor. Lentivirus titers were measured by p24 specific enzyme-linked immunosorbent assay.