Figure 2.

Restoration of H3K56 acetylation after UV damage repair is regulated by ASF1A histone chaperone. (A) ASF1A is required for H3K56Ac restoration in asynchronous NHF cells after UV damage repair. OSU-2 cells were transfected with ASF1A shRNA and after 24 h irradiated at 10 J m⁻² UV. Cultures were again transfected with ASF1A shRNA to maintain the knockdown up to 48 h post-irradiation. Whole cell lysates were prepared at the indicated times and H3K56Ac levels determined by western blotting. Asterik indicates cells treated with Fugene transfection reagent alone to exclude any possible toxicity effects on H3K56Ac levels. (B) Regulation of H3K56Ac restoration by ASF1A in response to UV irradiation is independent of the effects of ASF1A during replication. OSU-2 cells were arrested in the G1 phase of the cell-cycle by serum starvation for 48 h, transfected with ASF1A shRNA and exposed to 10 J m⁻² UV irradiation after 24 h of transfection. Cells were transfected again with ASF1A shRNA after UV irradiation to maintain the knockdown for a further 48 h. Cell lysates were prepared immediately or at the indicated post-UV repair times. H3K56Ac levels were determined by western blotting using anti-H3K56Ac antibody.