FIG. 6.

Viral genome copy number and cell viability in BC3 cells treated with drugs affecting PARP activity. BC3 cells were untreated (NT) or treated with NA or HU as described in Materials and Methods and tested for viral genome copy number by real-time PCR (A) and for cell viability by the trypan blue method (B). (A) Viral genome copy number in drug-treated cells. The viral genome copy number was estimated by amplifying the K-oriLyt CS region and normalized to the β-globin gene content by real-time PCR on the designated day. The number of copies is shown on a logarithmic scale as viral copy number per picogram of DNA. The value above the bar shows a mean value for each case. (B) Cell viability of dru-treated cells. The viable cells were counted on the designated day as described. (C) HU treatment leads to the increase in poly(ADP-ribosyl)ation of LANA. BC3 cells were treated with NT (nontreatment), TPA (25 ng/ml; Sigma), HU (50 μM), or NA (10 mM), and 3 days after treatment, cells were harvested and solubilized with RIPA buffer and the soluble fraction was immunoprecipitated (IP) with either a preimmune rat IgG or a rat monoclonal anti-LANA antibody. The immunoprecipitated fraction was fractionated by SDS-PAGE and immunoblotted (IB) with either rabbit polyclonal anti-PAR (αPAR) antibodies or a rat monoclonal anti-LANA (αLANA) antibody. (D) No lytic gene is expressed by drug treatment. BC3 cells were fixed with 4% paraformaldehyde-PBS 1 day after treatment with NT (nontreatment), TPA (25 ng/ml; Sigma), HU (50 μM), or NA (10 mM). Then the cells were incubated with a mouse monoclonal anti-RTA antibody, followed by goat anti-mouse IgG conjugated with Alexa 488 (Molecular Probes-Invitrogen) (green signal). The signal was detected with a confocal fluorescence microscope (Zeiss 510) (original magnification, ×600). The nucleus was counterstained with DAPI.