Loss of histone deacetylase 1 and 2 (Hdacs1,2) increases H4K16ac on nascent DNA and at replication origins. A-B. Bromodeoxyuridine (BrdU) pulse chase was performed to determine association of H4K16ac with nascent DNA at the indicated time points. C. BrdU-labeled nascent DNA associated with H4K16ac was measured by slot blot using input or immunoprecipitated DNA from cells transfected with non-targeting siRNA (NTsi) or Hdacs1,2-specific siRNA (H12si). BrdU-labeled nascent DNA was detected using an anti-BrdU antibody. Average BrdU signal of high and medium volume of chromatin immunoprecipitation (ChIP) DNA spotted on the slot was quantified by the Image J software and is shown on the right hand side of the panel. D-F. Changes in H4K16ac levels at candidate origins were measured by ChIP assays using NIH3T3 cells either serum-starved (SS, 0 h) or released into S-phase for the indicated time periods in the presence of either DMSO or 3.75 μM 898. Fold enrichment for H4K16ac was determined after normalization of H4K16ac occupancy to the occupancy of H4 at a given locus from three independent treatments.