1 of the Supporting Materials. Hematoxylin
and eosin (H&E) staining and immunohistochemistry staining of Ki67, 5-bromo-2-deoxyuridine (BrdU), proliferating cell nuclear antigen (PCNA), and p-H3S10 were performed. The number of positive nuclei per 1,000 cells was counted in consecutive high-power fields. Cell apoptosis was determined using a terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay according to the manufacturer’s instructions (Roche Applied Science, Switzerland). The ratio of apoptotic nuclei to the total nuclei was calculated. The mouse liver cancer cell line Hepa1-6 was maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine,
100 U/mL penicillin, and 100 μg/mL streptomycin. The cells were maintained at 37°C in a 5% (v/v) CO2 atmosphere and Tamoxifen price subcultured every 3 days. Transfection with siRNAs against the Hdac1, Hdac2, or Ki67 gene was performed using Lipofectamine2000 (for the nucleic acid sequences, see Table S.2). Scrambled siRNA was used as a control. Before transfection the cells were synchronized with 100 ng/mL nocodazole for 16 hours. At 48 hours after a 5-hour transfection, Hepa1-6 cells were trypsinized, washed with cold phosphate-buffered saline (PBS), and fixed in prechilled 70% ethanol at −20°C overnight. To analyze the cell cycle profiles, the cells were NVP-BKM120 stained with a propidium iodide solution, incubated at 37°C in the dark for 30 minutes, and examined Carnitine palmitoyltransferase II using a flow cytometer (Cytomics FC 500, Beckman Coulter, Brea, CA). To quantify the apoptotic
cells, the Annexin V-FITC Apoptosis Detection Kit (Beckman Coulter) was used according to the manufacturer’s instructions. The data were analyzed using MultiCycle AV software. The cells were seeded onto coverslips precoated with fibronectin. At 48 hours after transfection the cells were permeabilized in 0.1% Triton X-100 for 10 minutes. Immunofluorescence analysis was performed and the immunostained cells were visualized with a Bio-Rad A1S1 laser confocal microscope (Hercules, CA). A chromatin immunoprecipitation assay (ChIP) was performed using a Magna ChIP G Chromatin Immunoprecipitation Kit from Millipore (Billerica, MA) according to the manufacturer’s instructions. The sequences of primers for the Ki67 gene are presented in Table S.3 of the Supporting Materials. The results are expressed as the mean ± standard deviation (SD). The differences between groups were tested for significance using the unpaired, 1-tailed Student t test with Welch correction. P < 0.05 was considered significant. We generated hepatocyte-selective Hdac1−/−, Hdac2−/−, and Hdac1−/−,2−/− mice, and the genotypes of the mice were assessed using PCR analysis of tail DNA (Table S4; Fig. 1A,B). The hepatocytes of the Hdac1−/−, Hdac2−/−, and Hdac1−/−,2−/− mice displayed strongly reduced levels of HDAC1, HDAC2, and HDAC1,2 proteins, respectively (Fig. 1C,D).