Luminescence was measured using 50 μL of lysate with the GloMax® Multi-Microplate Multimode Reader (Promega) and normalized to that of the empty vector. The electrophoretic mobility shift assay (EMSA) was performed using the LightShift Chemiluminescent kit (Thermo Scientific) and 2-μg nuclear lysates and 1-ng biotinylated probes (sense-strand sequence: 5′-TTGAGGCCTACTTCAAAGACTGTGTG-3′). The biotinylated EBNA probes that were provided were used as INK 128 datasheet the negative control. Binding was performed at 37°C for 45 minutes in 20-μL reactions with EMSA buffer (12.5% glycerol, 0.5 mM of ethylenediaminetetraacetic acid, 0.3 mg of bovine serum albumin, 0.05% Nonidet

P40, and 1 μg of poly-dIdC). Also, 1 μL of PARP1 antibody (sc-74469X; Santa Cruz Biotechnology) was used. Streptavidin pull-down was performed with 10 μL of Dynabeads® M-280 streptavidin (Invitrogen), 1 μg of biotinylated EMSA probe, and 70-μg nuclear lysates in

100-μL reactions in EMSA buffer. Bound proteins were eluted by boiling and sent to the Protein and Proteomics Center in the National University of Singapore for matrix-assisted laser desorption/ionization time of flight (MALDI-TOF/TOF) analysis. DNA was extracted using the DNeasy Blood and Tissue kit (Qiagen, Hilda, Germany). Quantitative real-time polymerase chain reaction was performed using LightCycler® FastStart DNA MasterPLUS SYBR Green I (Roche, Basel, Switzerland) in 10-μL reactions containing 1 ng of total DNA. cccDNA was amplified with primers cccF and cccR (Supporting Table 1) and normalized to the relative amount of pcDNA3.1+, amplified BEZ235 purchase by primers pcDNA-F1 and pcDNA-R1 (Supporting Table 1). Histone H1 modification assay was performed with the PARP Universal Colorimetric Assay Kit selleckchem (R&D Systems) and 5-μg nuclear lysates. Also, 1-μL DNA duplexes (Supporting Table 2), formed by annealing equal amounts of 100-μM DNA oligomers, were used. Alkaline comet assays were performed with the CometAssay® kit (Trevigen, Gaithersburg, MD) and scored using TriTek CometScore™ version

1.5 software (TriTek Corporation, Sumerduck, VA). Annexin V staining was performed with Annexin V-Fluos (Roche). Apoptosis was measured using the Caspase-Glo® 3/7 Assay (Promega). The F-test for equal variance, followed by the one-tailed Student’s t-test with equal or unequal variance, were performed. To determine the host factors interacting specifically with the HBVCP that may be involved in transcriptional activation, DNA probes spanning the HBVCP were biotinylated and subjected to affinity pull-down assays. A strong band of approximately 120 kDa was selectively enriched from HepG2 nuclear lysate by the probe nt 1696-1722 of enhancer II23, 24 within the HBVCP (Fig. 1A). MALDI-TOF/TOF analysis revealed that the bound protein was PARP1 (Fig. 1A; Supporting Fig. 2).