Tlrl pma

The African green monkey kidney Vero E6 cells were purchased from ATCC (ATCC CRL-1587), ACE2-overexpressing A549 (ACE2-A549) cells were a gift from Dr. Olivier Schwartz (Institut Pasteur, France), ACE2 and transmembrane serine protease 2 (TMPRSS2)-expressing A549 (ACE2-TMPRSS2-A549) cells were from Invivogen (#a549-hace2tpsa) and HEK293T cells were from ATCC (#CRL-11268, #CRL3216). These cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) with 10% heat inactivated fetal bovine serum (FBS), 100 UI/ml penicillin (Life technology), and 100 μg/ml streptomycin (Life technology) at 37°C. Caco-2 cells were kindly provided by Pr. Guido Kroemer (Gustave Roussy, France) and were cultured in EMEM (30-2003, ATCC) with 20% heat inactivated FBS, 100 UI/ml penicillin, and 100 μg/ml streptomycin at 37°C. Monocyte THP1 cells (TIB2002, ATCC) were differentiated in macrophages with phorbol-12 myristate-13-acetate (PMA) (#tlrl-pma, Invivogen), as previously described (47 (link)) and maintained in RMPI-1640-Glutamax medium supplemented with 10% heat inactivated FBS, 100 UI/ml penicillin, and 100 μg/ml streptomycin. All cell lines used were mycoplasma-free.

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.

The African green monkey kidney Vero E6 cells were purchased from ATCC (ATCC CRL-1587), ACE2-overexpressing A549 (ACE2-A549) cells were a gift from Dr. Olivier Schwartz (Institut Pasteur, France), ACE2 and transmembrane serine protease 2 (TMPRSS2)-expressing A549 (ACE2-TMPRSS2-A549) cells were from Invivogen (#a549-hace2tpsa) and HEK293T cells were from ATCC (#CRL-11268, #CRL3216). These cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, USA) with 10% heat inactivated fetal bovine serum (FBS), 100 UI/ml penicillin (Life technology), and 100 μg/ml streptomycin (Life technology) at 37°C. Caco-2 cells were kindly provided by Pr. Guido Kroemer (Gustave Roussy, France) and were cultured in EMEM (30-2003, ATCC) with 20% heat inactivated FBS, 100 UI/ml penicillin, and 100 μg/ml streptomycin at 37°C. Monocyte THP1 cells (TIB2002, ATCC) were differentiated in macrophages with phorbol-12 myristate-13-acetate (PMA) (#tlrl-pma, Invivogen), as previously described (47 (link)) and maintained in RMPI-1640-Glutamax medium supplemented with 10% heat inactivated FBS, 100 UI/ml penicillin, and 100 μg/ml streptomycin. All cell lines used were mycoplasma-free.