A model to describe this phenomenon is provided in the Discussion. Open in a separate window Figure 6 Both bithionol and hexachlorophene inhibit BKV DNA replication in Vero monkey kidney cells. supernatants were removed. The pellets were resuspended in TCA sample buffer, and then separated by 12.5% SDS-PAGE. The proteins were visualized with Coomassie Brilliant Blue. 2.4 Viral replication LIN41 antibody and cell culture assays CV1 cells were grown in MEM + 10% FBS and pen/strep and Vero cells (kindly provided by Dr. Bruce McClane, University of Pittsburgh) were grown in DMEM + 10% FBS + pen/strep at 37C in a 5% CO2 incubator. SV40 stocks were prepared by plating CV1 cells into 24-well dishes and infecting the cells with SV40 at a multiplicity Polydatin (Piceid) of infection (MOI) of 2 for 2 h. Next, the media was removed and replaced with media containing the desired compound or an equivalent volume of DMSO. Two biological replicates corresponding to each treatment were performed. The media was refreshed at 24 hours post infection (hpi), and again supplemented with either DMSO or the indicated compound. At 48 hpi, when the viral replication cycle of SV40 is nearly complete, the cells were frozen and thawed 3 times to provide a viral stock. This stock was titred by plaque assay using at least 3 technical replicates, based on a previously reported protocol (Murata et al., 2008). In brief, CV1 cells were Polydatin (Piceid) grown on 6 cm dishes to near confluence and dilutions of the viral stock were plated onto the monolayer for 2 h, and then replaced by a 4mL overlay of media in 0.9% Noble agar. On 3 and 6 days post infection (dpi), an additional agar overlay was made. At 9 dpi, the agar was removed and the monolayer was stained with crystal viol et. Plaques were counted by eye, and viral mediated Polydatin (Piceid) cell clearing was confirmed by light microscope. A quantitative DNA replication assay for SV40 was performed as previously described (Huryn et al., 2011; Li and Kelly, 1984; Randhawa et al., 2005a). CV1 cells at ~90% confluency were infected with SV40 at an MOI of 6, and after a 2 h infection the media was removed and replaced with media containing the desired compound or an appropriate volume of DMSO. These growth conditions were used to mimic normal viral infection in non-dividing cells. The media containing the compound was refreshed daily and at 48 hpi the viral DNA was harvested by free-thawing the cells (at ?20 C) in media three times. The DNA from the resulting cell lysates was stored at ?20C and was then quantified by quantitative PCR (qPCR) as outlined below. To obtain larger quantities of SV40 DNA and to directly visualize the viral DNA (see Figure S2), CV1 cells were grown to 90% confluency in 10 cm culture dishes, and were infected with SV40 at an MOI of 6. After 2 h of infection, the virus-containing media was replaced with fresh media containing the appropriate drug concentrations or the appropriate volume of DMSO. Dilutions were prepared in DMSO for bisphenol and hexachlorophene and each dilution was examined in cells in triplicate. After 24 hpi, the media was replaced with fresh media containing the appropriate drug dilutions. At 48 hpi, the cells were collected and viral DNA was extracted using the modified Qiagen miniprep protocol (Cantalupo et al., 2005). Specifically, cells were washed in PBS and then 250 L of buffer P1 was added to each well. Next, 250 L of P2 was immediately added to lyse the cells. When cells were visibly lysed by examination by light Polydatin (Piceid) microscope, the lysate was Polydatin (Piceid) removed and incubated with 500 g Proteinase.