Cells were lysed with 1% NP40 NET buffer for total protein extraction. For subcellular protein fractionation, cells were first lysed with buffer A (10 mM of HEPES [pH 7.9], 10 mM of KCl, 0.1 mM of ethylenediaminetetraacetic acid [EDTA]

0.1 mM of ethylene glycol tetraactic acid [EGTA], 1 mM of dithiothreitol [DTT], and 0.5 mM of phenylmethylsulfonyl fluoride [PMSF]) for 15 minutes, followed by centrifugation at 12,000 rpm for 5 minutes. Cytosolic fraction was collected, and the pellet was lysed with buffer C (20 mM of HEPES [pH 7.9], 0.4 M of NaCl, 1 mM of EDTA, 1 mM of EGTA, 1 mM of DTT, and 1 mM of PMSF) for 15 minutes. Nuclear fraction was collected after centrifugation at 12,000 rpm for 5 minutes. Proteins AP24534 manufacturer were resolved in sodium dodecyl sulfate/polyacrylamide Talazoparib manufacturer gel electrophoresis and blotted onto nitrocellulose membrane. The membrane was incubated with primary antibody (Ab) overnight at 4°C, followed by antimouse or -rabbit immunoglobulin G (GE Healthcare, Waukesha, WI) for 1 hour at room temperature. The ECL detection system (GE Healthcare) was used for protein detection according to the manufacturer’s protocol. Anti-SUV39H1 and -topoisomerase

I (Topo I) Abs were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-α-tubulin and -β-actin Abs were purchased from Sigma-Aldrich (St. Louis, MO). Anti-H3K9me3 and -panH3 Abs were ordered from Abcam (Cambridge, MA) and Upstate Biotechnology (Lake Placid, NY), respectively. Clinicopathological features of HCC patients were

analyzed as previously described.18 Statistical analysis was performed using SPSS 19 for Windows (SPSS, Inc., Chicago, IL). Fisher’s exact test was used for categorical data, independent t test was used for continuous parametric data, and Mann-Whitney’s U test was used for continuous nonparametric data. For overexpression study, SUV39H1 expression vector (CMV-[myc]3-SUV39H1) was kindly provided by Prof. Thomas Jenuwein (Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany), and the full-length of SUV39H1 coding sequence was further 上海皓元医药股份有限公司 subcloned into pEGFP-C2 expression vector (Clontech Laboratories, Inc., Mountain View, CA). SUV39H1 was transfected into HCC cells using Lipofectamine 2000 (Invitrogen), and stably overexpressing cells were selected using 1 mg/mL of G418. For knockdown study, lentiviral delivery of short hairpin RNAs (shRNAs) targeting SUV39H1 (shSUV-1 and shSUV-2) and nontarget control (NTC) (Sigma-Aldrich) were used. Stably transduced cells were selected by puromycin at 1 µg/mL. HCC cells were seeded at 2 × 105 cells per well onto a six-well plate for transfection or viral transduction. Three days after transfection or viral transduction, 5% of the cells were seeded onto another six-well plate and subjected to G418 or puromycin selection for 2 weeks. Resistant colonies were fixed with 100% methanol and stained with crystal violet.