Sigenome non targeting sirna 3

WI38 cells were purchased from American Type Culture Collection. Cells were cultured in DMEM supplemented with 10% FBS (R&D Systems) and 1% Penicillin-Streptomycin (ThermoFisher Scientific). These cells were screened for mycoplasma every four months using the ATCC Universal Mycoplasma Detection Kit (Catalogue # 30–121 1012 K). Early passage primary MEFs were harvested and cultured from C57BL/6 mice as previously described [20 (link), 44 (link), 45 (link)]. β-gal staining was achieved using the Cellular Senescence Kit (Millipore). Recombinant proteins included Cyclophilin A (R&D) and S100A4 (Abcam). SN52 peptide (AAVALLPAVLLALLAPVQRKRRKALP) and SN52-mut peptide (AAVALLPAVLLALLAPVQRNGRKALP) were acquired form GenScript. Inhibitors used were: WP1066, BMS345541, G06976, Staurosporine, and KU60019 (all from Selleck), and Roscovitine (Sigma). The following siRNAs were used: siGENOME non-targeting siRNA#3 (Dharmacon) and si-STAT3 (sc-29,493, Santa Cruz). siRNA transfection was performed with Oligofectamine (Invitrogen).

HUVECs were grown to 90% confluency on 60-mm (2 (link)) gelatin-coated tissue culture plates and transfected with siRNA at a final concentration of 40 nM using Oligofectamine (Invitrogen) in a total volume of 2000 μl. Transfection occurred for 4 h at 37 °C in Opti-MEM medium (Invitrogen), after which M199 medium (Invitrogen) containing fetal bovine serum (Hyclone), heparin, and endothelial cell growth supplement (Biomedical Technologies) was added. Cells were incubated with siRNA for 24 to 48 h depending on the downstream application. Custom Stealth siRNAs (Invitrogen) were used to knockdown GATA2-AS1. SiGENOME Non-Targeting siRNA #3 (Dharmacon) was used as control siRNA transfection (Table S3).

U87, A172, and HEK293T (293T) cells were purchased from American Type Culture Collection. HeLa cells stably expressing empty vector (EV), 6His-SUMO 1 and 6His-SUMO 2, were obtained from Dr. RT Hay and have been described previously [37 (link)]. Cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin (ThermoFisher Scientific). GBM34 and GBM44 GSCs obtained from Dr. Mariano Viapiano (Brigham and Women’s Hospital, Boston, MA) were maintained as neurospheres as described [27 ]. All cell lines were screened for mycoplasma using the ATCC Universal Mycoplasma Detection Kit (catalogue # 30- 121 1012K) every 4 months. Early passage primary MEFs from wild-type, MyD88^−/−, Rig-I^−/−, Mda5^−/−, Lgp2^−/−, MAVS^−/−, Tmem173^−/−, and Trif^−/− mice were cultured as previously described [51 (link), 52 (link)]. All cell lines were authenticated by routine morphological and growth analysis and by western blotting. TMZ was obtained from Sigma-Aldrich. DNA transfection was performed using TransIT LT1 (Mirus) and siRNA transfection with Oligofectamine (Invitrogen). Cycloheximide (CHX) and MG132 were from Cayman Chemical. The following siRNAs were used: siGENOME non-targeting siRNA#3 (Dharmacon), siGENOME Smartpool si-LGP2 (Dharmacon), si-TLR3 (sc-36685, Santa Cruz), and si-PKR (sc-36263, Santa Cruz).