Delayed administration of NAC exhibits decreased levels of protection against AAP and CDDP-induced cytotoxicity. (A) Western blot analysis of HUH6 cells detecting cleaved PARP as a marker for apoptosis and tubulin to confirm equal protein loading. Five micrograms of CDDP per milliliter and/or 10 mM AAP was added at time 0, and 1 mg/ml NAC was added at times 0, 1, 2, and 4 hours. Cells were incubated in treated medium overnight. (B) Quantification of the immunoblot signal of cleaved PARP in panel A was performed from three independent experiments. Statistical significance between two treatment groups was indicated by asterisks: *P < .05 or **P < .01. (C) WST cell viability assay of HepT1 cells. All cells were treated with 5 µg/ml CDDP and 10 mM AAP at time 0, and then either no NAC, or 1 mg/ml NAC at 0, 2, or 6 hours (n = 3, mean ± SEM). Cells were incubated in treated medium overnight before being developed for analysis. Statistical significance between treatment and “No NAC” control group was indicated by asterisks: *P < .05 or **P < .01.