Separation of nuclear proteins by SDS-PAGE identified three
clearly visible bands with increased intensities in samples obtained
from FA-treated cells (with or without CoCl2; Fig. 5A).
Analysis of these bands using MS allowed to identify core histone
species H2A.1, H2B.1, H3.3 and H4. Although it was not identified
by MS, we investigated the levels of histone 2AX (H2AX) protein
and its phosphorylated form by Western blot analysis. H2AX is
H2A isoform which undergoes phosphorylation upon formation of
double strand DNA break, telomere dysfunction, interrupted replication/transcription,
virus infection, and apoptosis. Phosphorylated
H2AX foci serve as platforms for the recruitment of DNA
repair and chromatin remodeling factors as well as factors involved
in the cell-cycle checkpoint. Therefore, phosphorylated
H2AX is a useful marker of DNA damage [21]. Both the total as well
as the phosphorylated H2AX protein levels were enhance by FA treatment (Fig. 5B and C), and the ratio between phosphorylated
H2AX to total H2AX was considerably higher in these cells
(Fig. 5D). By contrast, CoCl2 had no significant effect on total or
H2AX phosphorylation.