NAC inhibits adaphostin-induced ROS production and cytotoxicity in K562 cells. (A) K562 cells were treated for 90 minutes with diluent (light solid line), 10 μM adaphostin (heavy solid line), or 10 μM adaphostin in the presence of 24 mM NAC (added 15 minutes prior to adaphostin; dotted line). Peroxide production was assessed using CM-H₂DCFDA. (B) K562 cells were treated for 0 to 90 minutes with 10 μM adaphostin and stained with CM-H₂DCFDA. Fluorescence intensity was measured as indicated in panel A and normalized to diluent-treated cells. (C) K562 cells were treated with 10 μM adaphostin for the indicated length of time. Whole-cell lysates were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by blotting with serum that recognizes the activating phosphorylation of Chk2 on Thr.⁶⁸ As a loading control, the same blot was probed with anti-Chk2 monoclonal antibody. (D) K562 cells were treated for 1 hour with diluent (heavy solid line), 10 μM adaphostin (light solid line), or 10 μM adaphostin after a 15-minute pretreatment with 50 μM zVAD (OMe)-fmk (dotted line). Superoxide was detected using H₂E. (E) K562 cells were treated for 24 hours with the indicated concentrations of adaphostin alone (○) or together with 24 mM NAC (•). At the completion of the incubation, cells were washed and plated in 0.3% agar to allow colonies to form. Error bars, ± SD from quadruplicate samples. (F) Peripheral blood granulocytes (left panel) or mononuclear cells (right panel) from a previously untreated patient with CML were incubated for 1.5 hours in diluent (light solid line), 10 μM adaphostin (heavy solid line), or 10 μM adaphostin added 15 minutes after 24 mM NAC (dotted line). At the completion of the incubation, cells were stained with CM-H₂DCFDA and examined by flow microfluorimetry to quantitate peroxide levels. Similar results were obtained in 2 additional CML patients.