Figure 2

Inhibition of histone deacetylase 1 and 2 activities within S-phase or their knockdown reduces replication fork velocity. A. Serum-starved NIH3T3 cells were released into S-phase in the presence of DMSO or 3 μM 898. Fluorescence-activated cell sorting analysis following bromodeoxyuridine (BrdU)-propidium iodide labeling and staining was performed at 0 h, 12 h, 18 h and 24 h following release into S-phase. B. Slot blot analysis was performed using the indicated amount of BrdU-labeled genomic DNA from DMSO or 3 μM 898 treated cells. BrdU incorporation was quantitated using densitometry. C. Fork velocity was measured by DNA combing. After 20 h following release into S-phase in the presence of DMSO or 3 μM 898, NIH3T3 cells were labeled with IdU (green) for 15 min and then with CldU (red) in the presence of 250 μM hydroxyurea for 20 min. DNA fibers were prepared and analyzed as described (Methods section). Box plots show average fork velocity of DNA fibers prepared from four independent 898 or 233 treatments. At least 100 fibers in different areas of the slide from three different slides were analyzed per experiment. D. Fork velocity was measured in NIH3T3 cells transfected with non-targeting (NT) or Hdacs1,2 (H12) siRNA. Box plots show average fork velocity from two independent experiments. The ‘box’ in each box plot (C-D) extends from the first to third quartile of the data, heavy dark line inside the box represents the median, and the ‘whiskers’ extend to data points no more than 1.5 times the interquartile range from the ends of the box. Open circles represent data points further from the ends of the box than 1.5 times the interquartile range. Two sided P values were determined using Welch’s two-sample t-test. R statistical computing software (version 2.15.0) was used to construct box plots and for t-tests.