Pyrazofurin

MRC-5 cells and the HCoV-229E strain were purchased from the American Type Culture Collection (ATCC; Manassas, VA, USA). All media ingredients and the XTT-based viability assay kit were purchased from Biological Industries (Beit HaEmek, Israel). Cannabidiol (CBD) was purchased from Recipharm Israel (Ness Ziona, Israel). NT-VRL-1 formulation was obtained from Eybna Technologies (Givat Hen, Israel). Glycyrrhizin was obtained from Penta International Corporation (New Jersey, NJ, USA) and pyrazofurin was purchased from Sigma (Jerusalem, Israel).

Thymidine, hypoxanthine, sodium formate, adenine, adenosine, guanosine, deoxyuridine, H₂O₂ and pyrazofurin are from Sigma Aldrich. AICAr is from Chemtronica. Febuxostat is from Stratech Scientific Ltd. Allopurinol and Uric acid were from Merck Life Science UK.

Drugs were spotted using a Tecan D300e dispenser (Tecan, Mannedorf, Switzerland) at the following concentrations with log2 dilution series. pyrazofurin (SML1502, Sigma-Aldrich) (200 μM–5 μM for U2OS and hTERT-RPE1; 200 μM–2 μM for MCF7), MPA (M5255, Sigma-Aldrich) (100 μM–0.05 μM), 6MP (38171, Merck) (100 μM–0.05 μM), glutamine starvation (2 mM-7.8 μM), brequinar (5.08321 Sigma-Aldrich) (200 μM–10 μM), and leflunomide (L5025, Sigma-Aldrich) (400 μM–10 μM), hydoxyurea (H8627, Sigma-Aldrich) (5 mM–0.05 mM). Five hundred cells per well were seeded on 384-well cell culture plates, and viability was assessed with a resazurin assay and read on a Hidex Sense or Molecular Devices ID5 plate reader after 4 days in technical triplicates. Uridine (U3750, Sigma-Aldrich) rescue was performed at a concentration of 500 μM for 24 h. 2-DG (10 mM) was added to cultured cells for 48 h. Doxorubicine (D1515, Sigma-Aldrich), actinomycin D (A1410, Sigma-Aldrich), olaparib (AZD2281, Selleckchem), camptothecin (C9911, Sigma-Aldrich), nutlin3a (SML0580, Sigma-Aldrich).

For cell growth curve, 1500 cells were plated in one well of 96-well plate with at least 3 repeats for each cell and Cell Counting Kit-8 (CCK-8, Dojindo, Tokyo, Japan) was used to do continuous detection for 4–5 days. For colony formation assay, 1000 cells were plated in one well of 6-well plate with at least 3 repeats for each cell and 7–9 days later were stained with crystal violet. Image J was used for colony number counting. For pyrazofurin (SML1502, Sigma-Aldrich, St. Louis, USA) affecting cell growth, 1500 cells were plated in one well of 96-well plate, with at least 3 repeats for each condition and the next day pyrazofurin or vehicle was added. CCK-8 was used to do the continuous detection for 4–5 days. For cell cycle, one million of cells were harvested and fixed using pre-cold 70% ethanol at 4 ℃, overnight. The next day, centrifuge and discard the supernatant, and wash the cells with PBS twice. Then add the propidium iodide (PI) staining solution (C1052, Beyotime Biotechnology, shanghai, China) and incubate cells at room temperature for 15 min. Then go for the flow cytometric analysis. For cell apoptosis, the DKC1 knockdown A549 and PC-9 cells and control cells were harvested for apoptosis markers (P21 and γH2A.X) analysis using western blot.