Cb 839

5x10³ cells of parental HT-29 or HT-29 transfected with an empty vector were seeded in a 6-well plate containing MEM medium supplement with 10% (v/v) fetal bovine serum and 1% penicillin-streptomycin. Following 24 h culture, the old medium was replaced with a fresh medium containing 0-7.7 mM of 5-FU (Sigma-Aldrich, St Louis, USA) or 0-1000 µM of CB-839 (MedChem Express (Monmouth Junction, NJ, USA) at 37°C for 48 h. Stock solutions of 5-FU and CB-839 were prepared by dissolving the drug in 0.2% (v/v) DMSO before being 10-fold diluted in a culture medium. The viability of cells exposed to different drug concentrations was determined by an MTS assay. Dose-response curve of the drug was plotted between absorbance at 540 nm and drug concentrations. Half-maximal inhibitory concentration (IC₅₀) was then calculated from the graph. The viability of PC KO HT-29 cells cultured in the absence or presence of drug was relative to that of the empty vector transfected-HT-29 cells (control cell line) cultured in MEM without drug, which was arbitrarily set to 100%. DMSO was also included in MEM at a final concentration of 0.02% (v/v) (ThermoFisher, USA) for the no-drug treatment group. Apoptotic profiles of cells were analyzed using InvitrogenTM Attune NxT Software (ThermoFisher, USA).

All human Burkitt lymphoma cell lines were purchased from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). MDA-MB-468 breast cancer cell line was purchased from American Type Culture Collection (ATCC). BL cell lines were grown in RPMI-1640 medium supplemented with heat-inactivated 10% or 20% (RAMOS) fetal bovine serum (FBS), 100 U/mL penicillin, 100 U/mL streptomycin, and 25 mmol/L HEPES buffer (all from Lonza) at a density of 0.5–2.0 × 10⁶ cells/mL. MDA-MB-468 cell line was cultured in DMEM low glucose medium (Lonza) supplemented with FBS, penicillin, and streptomycin as above. All cells were grown in a humidified atmosphere at 37 °C with 5% CO₂.
PHGDH inhibitor NCT-503 (N-(4,6-dimethylpyridin-2-yl)-4-(4-(trifluoromethyl)benzyl) piperazine-1-carbothioamide) and NCT-503 inactive control (N-(4,6-dimethylpyridin-2-yl)-4-(pyridin-4-yl)piperazine-1-carbothioamide), unable to inhibit PHGDH) were purchased from Sigma-Aldrich. Glutaminase inhibitor CB-839 was purchased from MedChemExpress. All compounds were diluted in sterile dimethyl sulfoxide (DMSO) to obtain 10 mM stocks, aliquoted, and stored in 2–8 °C according to manufacturer’s recommendations.