Figure 3.

H3K56 acetylation is required for the dephosphorylation of γ-H2AX. (A) Depletion of ASF1A results in the retention of γ-H2AX for a longer time at sites of UV damage. OSU-2 cells were transfected with ASF1A shRNA and after 24 h irradiated with 10 J m⁻² UV through micro-pore filters placed on the cell monolayers. Cells were recovered at indicated times and fixed with paraformaldehyde. Immunofluorescence was performed using anti-γ-H2AX antibody. Images shown are from a representative of multiple experiments. (B) Quantitation of γ-H2AX foci in ASF1A-depleted cells. Triplicate experiments were performed as described above in (A) and the number of cells containing γ-H2AX foci in shRNA-treated and -untreated cells were quantitated as follows. An average of 150 merged nuclear foci from at least five different microscopic fields were used for the quantitation of γ-H2AX foci for each experiment. The graph depicts the average number of cells containing γ-H2AX foci from the three independent experiments. The error bars show the mean standard deviation. (C) H3K56R mutation results in the retention of γ-H2AX foci at sites of UV damage. H1299-H3.1 and H1299-H3K56R cells were exposed to 10 J m⁻² of UV radiation through micro-pore filters. Cells were fixed at the indicated intervals and immunofluorescence was performed to detect γ-H2AX foci. Images shown are from a representative experiment. (D) Quantitation of γ-H2AX foci in H1299 cells. Quantitation of γ-H2AX foci in H1299-H3.1 and H1299-H3K56R cells was essentially performed as described in (B) from two independent experiments. (E) H3K56 acetylation-deficient cells are defective in γ-H2AX dephosphorylation. H1299-H3.1 and H1299-H3K56R cells were irradiated at 10 J m⁻² UV and whole cell lysates prepared at 4, 8, 24 and 48 h. Equal quantities of cell lysates were resolved on SDS–PAGE and the levels of γ-H2AX determined by western blotting. Total H2AX and β-ACTIN were used as loading controls.