2.2. Drug treatments in cell culture -

Glioma cells were seeded in 6-well tissue culture plates 24 h prior to drug treatment. Cells were treated for 4 h with 10-20 μM 4-hydroperoxy-CPA (4HC; Santa Cruz Biotechnology, Inc), as specified in each study, then washed once and replenished with fresh, drug-free media. Cells were collected in TRIzol (Life Technologies, Grand Island, NY) to isolate RNA 24, 48, or 72 h after initiating 4HC treatment. The concentration 4HC was based on the EC₅₀ value for 4HC cytotoxicity, 12.9 μM, measured in cultured GL261 cells in a 24 h growth inhibition assay (data not shown). The 4 h exposure time was chosen to mimic the exposure to active 4-hydroxy-CPA that occurs in mice in vivo, where CPA and 4-hydroxy-CPA show plasma half-lives of 20-40 min, and are essentially eliminated from circulation within 4 h [40, 41]. In some studies, GL261 cells were seeded in 150 mm culture dishes 24 h prior to treatment. Cells (70–80% confluent) were treated with 10 μM 4HC for 4 h, washed and then replenished with fresh culture medium (10 ml). Conditioned media from the 4HC-treated cells was collected 48 h later, passed through a 0.45 μm syringe filter to remove dead cells and debris, and then applied to fresh GL261 cells seeded in 6-well plates 24 h earlier. Cells were then treated with the conditioned media or with culture media containing 250 U/ml IFNβ1 (Recombinant Mouse IFNβ1, Cat. #581304, BioLegend, San Diego, CA) and collected in TRIzol for RNA isolation either 6, 12, 24, or 48 h later. In other experiments, GL261 cells or CT-2A cells were seeded in 6-well plates 24 h prior to treatment. Cells were treated for 4 h with 20 μM 4HC with or without anti-IFNAR1 antibody (10 μg/ml; clone MAR1-5A3, Cat. # BE0241, Bio-XCell, West Lebanon, NH). Cells were then washed and replenished with fresh, drug-free media containing 10 μg/ml anti-IFNAR1 antibody, then harvested in TRIzol for RNA isolation 44 h later.