Abolishing histone deacetylase 1 and 2 (Hdacs1,2) activities causes DNA damage and replication stress, but transcription of genes for replication factors is not affected. A. NIH3T3 cells were treated with either DMSO or 3 μM 898 or 233 for 48 h and immunofluorescence with anti-γH2AX antibody was performed to examine activation of DNA damage response. B. Immunofluorescence using anti-Rad51 antibody was performed on serum-starved NIH3T3 cells following a 20 h release into S-phase and treatment with DMSO or 3 μM 898 or 3 μM 233 in the presence of 500 μM HU. A representative image from multiple treatments is shown. C-D. HeLa cells were treated with 3 μM 898 or 233 in the presence of 500 μM hydroxyurea for 48 h prior to chromatin preparation. Western analysis of chromatin was done to determine changes in RPA32, modified form of RPA32 (RPA32 S4/S8 phosphorylation, p-RPA32) and p53. p, phosphorylated and slow migrating species of RPA32. H3 and TBP (TATA binding protein) serve as loading controls. E. Total RNA was isolated from NIH3T3 cells released into S-phase following serum starvation and treated for 20 h with either DMSO or 3 μM 898. RNA samples obtained from three independent DMSO- or 898-treated cells were subjected to sequencing using the Illumina Hiseq2000 sequencer. Relative reads for CTP synthetase (CTPS) and Cyb561 genes in control and treated samples are shown. F. Chromatin was prepared from non-targeting (NT) or Hdacs1,2 (H12) siRNA transfected cells at 72 h time point for western analyses. H3 and H4 serve as loading controls. G. Serum-starved NIH3T3 cells were released into S-phase in the presence of DMSO or 3 μM 898. Chromatin extracts were prepared at 0 h (SS, serum-starved), 12 h, 18 h and 24 h following release for western analyses.