Figure 4.

Acetylation of H3K56 is not required for NER. (A) Depletion of ASF1A does not affect CPD repair efficiency. OSU-2 cells were transfected with ASF1A shRNA and irradiated with 10 J m⁻² UV after 24 h through micropore filters placed on the cell monolayers. Cells were fixed with paraformaldehyde at the indicated times and immunofluorescence was performed using anti-CPD antibody. Images shown are from a representative of duplicate experiments. (B) Quantitation of CPD foci in ASF1A-depleted cells. Duplicate experiments were performed as described above in (A) and the number of cells containing CPD 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 CPD foci for each experiment. The graph depicts the average number of cells containing CPD foci from two independent experiments. (C) ASF1A-depleted and H1299-H3K56R cells are proficient in NER. ASF1A shRNA-treated cells and H1299-H3K56R cells were irradiated at 10 J m⁻² UV and DNA isolated at the indicated post-irradiation times. Equal amounts of DNA was blotted on nitrocellulose membranes and probed for the presence of CPD and 6-4PP photoproducts with specific antibodies.