FIG. 2.

Analysis of histone H3 glutathionylation during cell proliferation. Glutathionylation of histone H3 increases during cell proliferation. (A) DNA synthesis rates for control (nontreated) and BioGEE (250 μM) treated NIH3T3 fibroblasts determined by BrdU incorporation assay (top). Immunoblot analysis of PCNA protein expression at 6, 24, 48, and 120 h of cell culture using total protein extracts from control and BioGEE treated NIH3T3 fibroblasts (bottom). Equal loading was controlled using anti-tubulin antibody in the immunoblots. The statistical significance is expressed as *p<0.05 between compared groups. (B) Changes in biotin-glutathionylated nuclear proteins during cell proliferation. Coomassie blue staining of SDS-PAGE gel containing nuclear proteins from Bio-GEE treated NIH3T3 fibroblasts harvested at different culture time points, used as loading control (top). Immunoblot using streptavidin-HRP to detect nuclear glutathionylated proteins (bottom). Notice a 15 kDa band corresponding to the size of histone H3 is observed (see arrow). (C) Analysis of glutathionylated proteins and H3 expression during NIH3T3 proliferation. Immunoblots of nuclear extracts from NIH3T3 cells incubated with either, anti-GSH or anti-H3 antibodies under nonreducing (left) and reducing conditions (right). (D) Free cellular NAD⁺/NADH ratios during cell cycle in NIH3T3 cells as cellular redox marker. The statistical significance is expressed as *p<0.05 between compared groups. GSH, glutathione; PCNA, proliferating cell nuclear antigen.