Figure 3. H2AX-diminished quiescent cell-status is abolished by continuous growth stimulation with accompanying H2AX recovery.

A. Quiescent MEFs with diminished H2AX expression were cultured under tSD-3T3 conditions until P10. They were then exposed to complete medium, which was changed every three days for 30 days. Immortal passages were started under Std-3T3 conditions (red circles). MEFs cultured under the Std-3T3 conditions (black circles) as in Figure 1a were superimposed for comparison of the time needed to acquire immortality. Representative images of MEFs during the process of acquiring immortality are also shown. B,C. Tetraploidy development in immortalized MEFs (IP2) was observed by flow-cytometry (B) and Giemsa staining (C). D. Growth acceleration-associated cell cycle progression and H2AX induction. To determine the effect of serum induction on H2AX expression and cell cycle progression, senescent MEFs at P8 were incubated in serum-free medium for 24 h and harvested after exposure to complete medium for various times. H2AX expression increased with increasing PCNA and histone H3, which suggests that the expression of these chromatin factors was associated with S phase entry. To detect H2AX levels in these MEFs at P8, the H2AX signal was visualized by longer exposure. E. DNA lesions characterized by γH2AX foci were induced in MEFs (red arrowheads) after exposure to complete medium as in D. F,G. H2AX status in immortalized MEFs was determined by Western blotting (F) and immunofluorescence (G), revealing H2AX recovery. H. DNA replication stress-associated H2AX diminution was compared between normal and immortalized MEFs as in Figure 2E, in which H2AX was not down-regulated after immortalization.