Tlrl patc

THP1-Dual or A549-Dual cells were seeded in 96-well plates and infected with 1 PFU/mL MOI of either LCMV-Arm or LCMV-WE, or treated with 100 ng/mL of the RIG-I-Like-Receptor (RLR) agonist Poly(dA:dT) pre-complexed with LyoVec transfection reagent (tlrl-patc, InvivoGen, San Diego, CA, USA) as a positive control for IRF reporter activation, or 1 ng/mL for THP1-Dual or 300 ng/mL for A549-Dual of the TLR2 agonist PAM3CSK4 as a positive control for NFκB activation. Forty-eight hours after treatment, supernatants were harvested and NFκB and IRF reporter activation were quantified on a Cytation 7 absorbance reader and luminometer (Agilent, Santa Clara, CA, USA) using Quanti-Blue and Quanti-Luc reporter systems according to the manufacturer’s directions (InvivoGen, San Diego, CA, USA). For NF-κB activation, the levels of SEAP were determined by reading the optical density at 655 nm. For IRF activation, the levels of Lucia luciferase were determined by measuring the relative light. Fold changes compared to uninfected cells are reported to standardize between experiments.

Human THP-1, U937, HeLa cells, A549, HCT-116, and mouse TRAMP-C2 cells were purchased from ATCC (USA). A549, HeLa and TRAMP-C2 cells were grown in DMEM medium (Invitrogen, USA), while HCT116 cells were cultured in McCoy’s 5A medium (Invitrogen, USA), and both mediums are supplemented with 10% heat-inactivated FCS (Hyclone, USA) and 1% pen/strep (Invitrogen). THP-1 and U937 cells were cultured in RPMI medium (Hyclone, USA) supplemented with 10% heat-inactivated FCS (Hyclone, USA). All cells were grown at 37°C in a humidified 5% CO2 incubator (Thermo Scientific, USA). THP1 cells were differerentiated in PMA (#tlrl-pma, InvivoGen, USA) and stimulated with 1 μg/ml LPS (#tlrl-peklps, InvivoGen, USA) overnight before fresh cell medium was replaced and the cells were either treated with 50% cancer CM or transfected with 1 μg/ml poly (dA:dT) (#tlrl-patc, InvivoGen, USA) with lyovec transfection reagent. Inhibitors THP1 Cells were primed with 100 ng/ml PMA, followed by LPS (1 μg/ml) for 3 hours prior to treatment 1 hour with 1 μg/ml Poly(dA:dT) complexes (AIM2 inflammasome inducer) or A549 tumor CM in the presence of ODN TTAGGG (A151; AIM2 inhibitor, 0.5 μM) or MCC950 (NLRP3 inhibitor, 0.5 μM). Cell supernatants were detected using ELISA.