6 well tissue culture treated plates

For 2D growth, cells were seeded (3 × 10⁵ cells per well) in tissue culture-treated 6-well plates (Costar). At the indicated time, cells were enumerated and stained with Trypan blue (ViCell XR, Beckman). Alternatively, cell metabolic activity was measured by a colorimetric XTT assay (Sigma). For 3D tumorspheres, cells were seeded at 10,000 cells/ml equivalent in poly-HEMA-coated 6-, 24-, or 96-well plates (Costar) for 5 days. At the indicated times, 3D tumorspheres were phase-contrast imaged (Olympus CKX41), enumerated (ViCell XR), or collected by centrifugation. Spheroid size was determined using Image J (NIH). Alternatively, cell metabolic activity was measured by a colorimetric XTT assay (Sigma). For methylcellulose colony formation, cells were suspended in 1% methylcellulose diluted in 2D growth media, 10⁴ plated in six-well poly-HEMA-coated plates, and colony formation analyzed after 21 days. Cells from methylcellulose colonies were collected by dilution-dispersion in PBS, centrifugation at 400 xg, and washed in PBS prior to enumeration or cell lysis. Cells were used at passage 10 to 35 and mycoplasma testing was performed every 3 months. For all experiments, triplicate experimental points were evaluated (technical replicates) and experiments were repeated at least two times (biological replicates).

Murine C₃H₁₀T_1/2 cells (ATCC) were plated in 6-well tissue culture treated plates (Corning Costar) at a density of 3 × 10⁵ cells/well in cell culture medium (88% DMEM, 10% heat-inactivated FBS, 1% 0.5 mM L-glutamine, 1% Penicillin/Streptomycin,) for 24 h. After 24 h, culture medium was replaced with MEM Alpha 1X (Gibco) and cells were transfected using Lipofecatime RNAiMax reagent (ThermoFisher Scientific) with either 30 pM of Scleraxis siRNA Catalog #sc-153257 (Santa Cruz) or 30 pM of Scrambled Control siRNA (FITC Conjugate)-A Catalog #sc-36869 (Santa Cruz) for 24 h. Cells were then lysed with RIPA Buffer for western blot analysis or TRIzol reagent for qPCR. Western blot data n = 3 and qPCR data n = 6. “n” denotes number of biological replicates.

Bone marrow-derived macrophages were seeded in 24-well or 6-well tissue culture treated plates (Costar) and allowed to adhere overnight at 37°C in 5% CO₂. Assay solutions were freshly prepared. Cells were washed and indicated wells were primed with 100 ng/mL E. coli 011:B4 LPS (Sigma-Aldrich) for three hours. After three hours, all wells were aspirated and replenished with fresh media, media containing 5 mM ATP (MPBiomedicals), or media containing live B. pertussis diluted from overnight cultures. Prior to collection of supernatant, plates were centrifuged at 500×g for 5 minutes.

Cells were seeded at a density of 10⁶ cells per well in 6-well tissue culture treated plates (Corning) and maintained at 37°C for 16 hours. Cells were then treated with NEU by direct addition of drug to the culture medium for 2 hours (unless otherwise indicated). Control cells were treated with equivalent volume of DMSO (drug solvent). For ATM and DNA-PK inhibition, cells were treated with 10 μM KU 55933 and 25 μM DMNB, respectively, immediately prior to addition of drug while for ATR inhibition 10 μM VE 821 was added one hour prior to addition of NEU to the cells. For time course studies, cells were treated with NEU for different time periods ranging from 0 to 120 minutes. After drug treatment, medium containing NEU was aspirated and cells were washed once with 1X phosphate buffered saline (PBS; PAN-Biotech GmbH). Cells were lysed in sample buffer containing 0.06 mM Tris (pH 6.8), 6% glycerol, 2% sodium dodecyl sulphate (SDS), 0.1 M dithiothreitol (DTT) and 0.006% bromophenol blue and lysates were stored at - 40°C.