One solution cell proliferation assay kit

A One Solution Cell Proliferation Assay kit (the MTS/phenazine methosulphate [PMS] method; Promega, Madison, WI, USA) was used to assess cell proliferation according to the manufacturer's instructions. After incubation for 4 h at 37°C, the absorbance was measured using an automatic microplate reader (Gene Company, Hong Kong, China) at an optical density of 490 nm (OD₄₉₀).

Maduramicin ammonium (molecular weight = 934.16, purity>97%, by HPLC) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution (5 mg/ml), aliquoted and stored at −80°C. Dulbecco's modified Eagle's medium (DMEM) and 0.05% trypsin-EDTA were obtained from Mediatech (Manassas, VA, USA). Fetal bovine serum (FBS) was from Atlanta Biologicals (Lawrenceville, GA, USA). One Solution Cell Proliferation Assay Kit was from Promega (Madison, WI). Cellular DNA Flow Cytometric Analysis Kit was purchased from Roche Diagnostics (Indianapolis, IN, USA). CF488A-Annexin V and Propidium Iodide (PI) Apoptosis Assay Kit was purchased from Biotium (Hayward, CA, USA). Enhanced chemiluminescence solution was from Perkin-Elmer Life Science (Boston, MA, USA). The following antibodies were used: cyclin A, cyclin B1, cyclin D1, cyclin E, CDK1, CDK2, CDK4, CDK6, CDC25A, CDC25B, CDC25C, p21^Cip1, p27^Kip1, Rb, p-Rb (S807/811), survivin, Mcl-1, Bcl-2, Bcl-xL, BAX, BAK, BAD, FasL, Fas/CD95, TNFα, TNFR1, TRAIL, DR4, DR5, FLIP S/L, FADD, TRADD, RIP (Santa Cruz Biotechnology, Santa Cruz, CA, USA), cleaved caspase 3, cleaved PARP (Cell Signaling, Beverly, MA, USA), β-tubulin (Sigma, St Louis, MO), goat anti-mouse IgG-horseradish peroxidase, and goat anti-rabbit IgG-horseradish peroxidase (Pierce, Rockford, IL, USA).

Cells seeded in 96-well plates were cultured overnight to form a confluent monolayer. For 10 mg/mL stock, ribavirin (Millipore, United States) was dissolved in phosphate-buffered saline (PBS). The culture medium was removed and the cells were subsequently incubated with ribavirin at concentrations range of 0 to 300 μg/mL in fresh culture medium. After 48 h of incubation, cell viability was evaluated using One Solution Cell Proliferation Assay kit containing MTS according to the manufacture^’s instructions (Promega, United States). The absorbance at 490 nm was read on a Synergy 2 Multi-Mode Microplate Reader (BioTek, United States).