Bptes

MEF WT p53^+/+, MEF p53^−/−, SaOs2-tetracycline inducible p53, E1A-RAS transformed MEFs, HT1080, EB3, 293T, CA46, SupT1, MiaPaca2, and 249P cells were initially cultured in Dulbecco’s modified Eagle’s medium (DMEM, Corning, New York, NY, USA) which contained 25 mM glucose and 4 mM L-glutamine. DMEM was supplemented with 10% dialyzed fetal bovine serum (dFBS, Gemini Bio-Products, Sacramento, CA, USA) and 100 units/mL penicillin and 100 μg/mL streptomycin (P/S, Genesee Scientific). All cells were cultured at 37° C with 5% CO₂. SaOs2 cells engineered to induce WT p53 upon tetracycline were generously provided by Karen Vousden (Francis Crick Institute, London, England) (Bensaad et al., 2006 (link)). Doxycycline (1 μg/mL) was added to DMEM 24 hours prior to glutamine deprivation studies to ensure p53 expression. BPTES (used at 10 μM) and camptothecin (CPT, used at 2 μM) was purchased from Sigma-Aldrich (St Louis, MO, USA). Doxorubicin was purchased from Selleckchem (Houston, TX, USA). Cell viability and proliferation was measured by exclusion of Trypan blue dye (Sigma) and determined by cell counting.

PARP antibody was purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA), goat anti-mouse and goat anti-rabbit HRP-conjugated IgG from Bio-Rad (Hercules, CA), anti-nuclear factor erythroid related factor 2 (NRF2) antibody from Abcam (Cambridge, MA) and ECL Plus solution from Thermo-Fisher Scientific (Waltham MA, USA). CB-839 was supplied by Calithera, Inc. (South San Francisco, CA) and was dissolved either in DMSO for in vitro experiments (10 mM stock, stored at -20 ⁰C) or in vehicle for in vivo experiments. The in vivo vehicle consisted of 25% (w/v) hydroxypropyl-β-cyclodextrin in 10 mmol/L citrate (pH 2). The GLS inhibitor BPTES (bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide) and all other reagents were purchased from Sigma (St. Louis, MO)

100 μL cells were added to flat bottom 96-well plates. After washing with warm PBS, monocytes were incubated with culture medium only as a negative control or 5 μg/mL of β-glucan, (β-1,3-(D)-glucan, kindly provided by Professor David Williams), 100 μM mono methyl-fumarate, or dimethyl-2-oxoglutarate (Sigma) for 24 hr (in 10% pooled human serum). Cells were washed once with 200 μL warm PBS and incubated for 5 days in culture medium with 10% serum and medium was changed once. Cells were restimulated with 200 μL RPMI, Escherichia coli LPS (serotype 055:B5, Sigma-Aldrich, 10 ng/mL), or Pam3Cys (EMC Microcollections, L2000, 10 μg/mL). After 24 hr, supernatants were collected and stored at −20°C (see Figure S1). In some experiments, cells were preincubated (before β-glucan training) for 1 hr with 10 nM rapamycin (Sigma), 50 μM BPTES (Sigma), 2 μg/mL Ceruline (Sigma), 100 μM 6-aminonicotinamide (Sigma), and 20 μM fluvastatin sodium hydrate (Sigma). Concentrations were selected as being the highest non-cytotoxic concentrations.