An antioxidant attenuates hyperacetylation of histone-H3 by NPI-0052 and HDACi in cell lines and primary cells. (A) NAC decreases histone-H3 expression and hyperacetylation of histone-H3 in Jurkat cells treated with NPI-0052 and MS-275. Cells were pretreated with 24 mM NAC for 30 minutes followed with 5 μM MS-275, 10 nM NPI-0052, or combination treatment for 6 hours. Cells were harvested, lysed, and analyzed by Western blot analysis for expression of Ac-H3, histone-H3, and actin. (B) Increased histone-H3 expression and hyperacetylation as a result of NPI-0052/MS-275 interaction is decreased by NAC in CLL patient sample. Peripheral blood from patient material (patient no. 1) was separated by centrifugation with a Ficoll gradient. Isolated cells were exposed to indicated doses of NPI-0052 and MS-275 for 6 hours after exposure to 24 mM NAC. Cell lysates were analyzed for Ac-H3, histone-H3, and actin expression by Western blot analysis. (C) Hyperacetylation of histone-H3 and increased histone-H3 expression by NPI-0052/vorinostat in CLL primary cells were attenuated with NAC. Mononuclear cells were isolated from CLL patient peripheral blood (patient no. 2). Cells were treated for 6 hours with 500 nM vorinostat, 10 nM NPI-0052, or a combination after exposure to 24 mM NAC for 30 minutes. Protein lysates were prepared and Western blot analyzed expression for Ac-H3, histone-H3, and actin. (D) Supplementation with glutathione ethyl ester (GSH-EE) reduces hyperacetylation of histone-H3 by NPI-0052 in primary cells. Isolated CLL primary cells (patient no. 3) were pretreated with 2 mM GSH-EE for 1 hour, followed by exposure to 10 nM NPI-0052 or diluent for 6 hours. Protein lysates were prepared and analyzed by Western blot analysis for Ac-H3, histone-H3, and actin expression.