5 fluorouracil

MARC-145 cells (ATCC) were cultured in Modified Eagle Medium (MEM; Sigma-Aldrich, St. Louis, MO, United States) containing 10% fetal bovine serum (FBS; Sigma-Aldrich, St. Louis, MO, United States) and 1% penicillin–streptomycin (Thermo Fisher Scientific, Waltham, MA, United States). BHK-21 cells purchased from ATCC were maintained in Dulbecco’s Modified Eagle Medium supplemented with 10% FBS (Gemini Bio, West Sacramento, CA, United States) and 1% penicillin–streptomycin (Thermo Fisher Scientific, Waltham, MA, United States). The HP-PRRSV TA-12 strain (GenBank Accession No. HQ416720.1) was purified by plaque assay in MARC-145 cells, and the complete genome of one plaque was determined by the Sanger sequencing method and deposited to GenBank under accession No. MZ399801. A monoclonal antibody (clone 4A5) against PRRSV N protein was purchased from MEDIAN Diagnostics, Korea. IFN-α2b (Sangon Biotech, Shanghai, China), ribavirin (Sigma-Aldrich, St. Louis, MO, United States), 5-Fluorouracil (MedChemExpress, Shanghai, China), and Chloroquine (MedChemExpress, Shanghai, China) were used in this study. Coelenterazine h (Maokang Biotechnology, Shanghai, China) was dissolved in acidified methanol to a concentration of 5 mg/ml, and aliquots were stored at −80°C.

The cell viability, colony formation, cell cycle, cell migration, and invasion assays were performed as described previously [20 (link)]. PI3K inhibitor GDC-0941 was purchased from ApexBio (Shanghai, China) [8 (link)], 5-Fluorouracil and paclitaxel were purchased from Med Chem Express (Shanghai, China).

Human melanoma cell line A375-M6, breast carcinoma MCF7 cell line, and colorectal carcinoma HCT116 cell line were maintained in DMEM 4,5 g/L glucose and 2 mM L-glutamine supplemented with 10% fetal bovine serum (Euroclone, Milan, Italy) as previously described¹⁰ (link). CAIX inhibitor SLC-0111, developed in the laboratory of Professor Claudiu T. Supuran (NEUROFARBA Department, University of Florence, Italy) and previously described¹⁰ (link), was used at 100 µM dose alone or in combination with Dacarbazine (Sigma Aldrich, Saint Louis, Missouri, USA) and Temozolomide (MedChemExpress, Sollentuna, Sweden) for melanoma cells, Doxorbicin (MedChemExpress) for breast cancer cells, or 5-Fluorouracil (MedChemExpress) for colorectal cancer cells, to evaluate a potential enhanced response of tumor cells to conventional chemotherapeutics. Dacarbazine, Temozolomide, Doxorubicin, and 5-Fluorouralcil were used at a lower dose than the half maximal inhibitory concentration (IC₅₀) (data not shown).

SHK was purchased from Sigma-Aldrich (St. Louis, MO, USA), dissolved in DMSO (Sigma-Aldrich, St. Louis, MO, USA). ZVAD-FMK, Necrostatin-1, 5-Fluorouracil and Oxaliplatin were from MedChemExpress (NJ, USA). ZDEVD-FMK and ZLEHD-FMK were from Adooq bioscience (CA, USA). N-acetyl-L-cysteine and L-glutathione were from Beyotime Institute (Shanghai, China). Antibodies were as follows: PARP, Caspase 3, Caspase 8, Caspase 9, RIPK1, Cytochrome C and VDAC1 were from Cell Signaling Technology (MA, USA); AIF, Endonuclease G, GAPDH and PCNA were from Abcam (Cambridge, UK); the goat IgG-HRP secondary antibodies against rabbit and mouse were from Zhongshan Golden Bridge Biotechnology (Peking, China) and FITC or Cy3 conjugated secondary antibodies were from Sigma (MO, USA).