Equafetal bovine serum

miR-2110 mimic, siRNAs and negative control oligos were purchased from Dharmacon. Rabbit anti-βIII-tubulin, anti-GAP43, anti-NSE, anti-TSKU, anti-calnexin, and HRP-conjugated anti-rabbit IgG antibodies were obtained from Thermo Fisher Scientific. Anti-cleaved PARP was purchased from Cell Signaling Technology. BE(2)-C, SKNDZ, CHLA-90, and SKNFI cells were from the American Type Culture Collection (ATCC). Kelly cells were obtained from the cell line repository at the Greehey Children’s Cancer Research Institute at the University of Texas Health San Antonio. Cells were grown in DMEM/F12 (Corning Cellgro) supplemented with 10% Equafetal bovine serum (Atlas Biologicals).

All cell lines were maintained at 37 °C, 5% CO₂. HeLa cells (ATCC (Manassas, VA, USA) CCL-2) were cultured in DMEM (Corning, Corning, NY, USA) supplemented with 10% Equafetal bovine serum (Atlas Biologicals, Fort Collins, CO, USA) and 1× l-glutamine (D10). 3T3 cells (ATCC, CRL-1658) were cultured in D10 supplemented with 1 mM sodium pyruvate (Corning) and 1× non-essential amino acids (GE Healthcare, Pittsburgh, PA, USA). NK92MI cells (ATCC, CRL-2408) were cultured in Alpha MEM (GE Healthcare) supplemented with 10% Equafetal bovine serum, 1× l-glutamine, 0.2 mM myo-inositol, and 0.02 mM folic acid. Caspase 1/11^−/− macrophages were isolated from C57BL/6 mouse bone marrow and cultured as previously described [4 (link)]. BMDM were differentiated for 7–21 days prior to experiments in DMEM that was supplemented with 30% L929 cell supernatant, 20% premium fetal calf serum (VWR Seradigm, Radnor, PA, USA), 1 mM sodium pyruvate, and 1× l-glutamine.

All cells were cultured in a humidified incubator at 37 °C with 5% CO2. Vector control and GSDMD KD THP-1 cells were generated by lentiviral shRNA-mediated targeting and were generously provided by Dr. Amal Amer at The Ohio State University. Cells were maintained in RPMI 1640 medium (Fisher Scientific) supplemented with 10% EquaFETAL bovine serum (Atlas Biologicals). THP-1 were differentiated into macrophages by treatment with 25 nM phorbol myristate acetate (PMA) for 3 days as previously described^{68 (link)} to allow for differentiation into macrophages. Infection of THP-1 cells with PR8 or H3N2 viruses was done at an MOI of 10 for 48 h prior to collection of cells for Western blotting or cellular media for measurement of cytokines and LDH release. MDCK cells (BEI Resources, NR-2628) were grown in DMEM (Fisher Scientific) supplemented with 10% EquaFETAL bovine serum.

GBM cell lines, primary GBM cells, and normal human astrocytes were cultured as described previously (7 (link), 14 (link), 47 (link)). In brief, GBM cell lines A172, LN-18, SF-268, SF-295, T98MG, U251, and U87MG were maintained in Dulbecco's Modified Eagle Medium (DMEM, Life Technologies) supplemented with 10% EquaFETAL® bovine serum (Atlas Biologicals, Inc.) and 100 μg/ml streptomycin and 100 IU/ml penicillin (Gibco). Primary cells VTC-001, VTC-002, VTC-004, VTC-037, VTC-056, VTC-058, VTC-084, and VTC-103 were cultured in DMEM supplemented with 15% fetal bovine serum (Peak Serum, Inc.) and penicillin/streptomycin. Normal human astrocytes were cultured in MCDB-131 Medium (Sigma) containing 3% fetal bovine serum (Peak Serum, Inc.), 10 X G-5 Supplement (Gibco), and penicillin/streptomycin. Cell lines have been authenticated by the ATCC authentication service utilizing Short Tandem Repeat (STR) profiling. Primary GBM cells were kept at low passages (no more than 10). Antibodies of RAN and GAPDH were purchased from Santa Cruz Biotechnology, Inc. Importazole was purchased from Cayman Chemicals, Inc. Stock solution of importazole was prepared at 50 mM using dimethyl sulfoxide (DMSO). Working solution was further diluted using cell culture media.

BL41, Ramos, Mutu I and BL8 cells were cultured in ‘complete’ RPMI 1640 medium (Cytiva, Marlborough, MA) containing 10% Equafetal bovine serum (Atlas Biologicals, Fort Collins, CO) 100 μg/mL penicillin-streptomycin and 2 mM glutamax (ThermoFisher, Waltham, MA) at 37°C and 5% CO₂. Cells were treated withO-tetradecanoylphorbol-13-acetate (TPA) at 25 ng/mL and sodium butyrate (NaBu) at 4 mM, TPA solubilized in EtOH/acetone and NaBu solubilized in H₂0, or 60 μM hemin (Sigma Aldrich, St. Louis, MO) solubilized in 1.4 M NaOH and compared to mock (NaOH added to complete media only) to measure viral and cellular changes.

HeLa cells (ATCC CCL-2) were maintained at 37 °C, 5% CO₂ in DMEM (Corning, Corning, NY, USA) supplemented with 10% Equafetal bovine serum (Atlas Biologicals, Fort Collins, CO, USA) and 1 × L-glutamine (D10). They were negative for Mycoplasma by microscopy.