Human Cytomegalovirus UL114 Interact with SMARCB1 Figure 4. Increased expression of the SWI/SNF core subunits in HCMV infected cells. (A) The expression of UL114, SMARCB1 and UL44 in nuclear extracts (20 mg) from mock and HCMV-infected fibroblast cells was analyzed at immediate –early (12 hpi), early (24 hpi) and late (48 and 72 hpi) times of infection by western blot. The western blots were analyzed by Thyphoon scanning. GAPDH was used as a loading control. (B) The expression of BRG1, BAF155 and BAF 170 in nuclear extracts (40 mg) from mock and HCMV-infected fibroblast cells was analyzed at late (72 hpi) time of infection by western blot. The western blots were analyzed by Thyphoon scanning. GAPDH was used as a loading control. doi:10.1371/journal.pone.0034119.g004 (MEM)+Dulbecco’s modification of Eagle’s medium (DMEM) (Gibco, LifeTechnologies Ltd. supplemented with endotoxin-free fetal calf serum (FCS), L-glutamine (0.3 mg/ml), gentamicin (40 mg/ml), amphotericin B (Fungizone) (2.5 mg/ml), and penicil- lin G (6 mg/ml). Medium with 10% FCS was used for propagation of the cells whereas medium with 2% FCS was used for maintenance of the cells. The HE-cells were routinely screened for mycoplasma by DNA staining with MycoAlert Mycoplasma Detection kit (Lonza Rockland Inc.). Stocks of highly purified human cytomegalovirus (HCMV) laboratory type strain AD169 (American Type Culture Collection (ATCC), Rockville, MD) was propagated in low passage HE cells. Virus was propagated at low virus to cell ratios to minimize generation of defective particles and purified as described previously [66]. Cell cycle synchronization and infections All cell experiments were performed subsequent to release from contact inhibition. The cells were grown to confluence, and after three days of confluence, they were trypsinized and re-plated at a lower density (106cells/75 cm2) to induce progression into the cell cycle. At the time of re-plating (1:1 MEM+DMEM-10% FCS), the cells were infected with HCMV at a multiplicity of infection (moi) of 5 plaque forming unit (PFU) per cell or mock infected. Virus adsorption was allowed for one hour before the medium was discarded and fresh medium (1:1 MEM+DMEM-10% FCS) was added. Only experiments where more than 95% of the cells were infected were accepted. The infection was assessed at 24 hpi by an immunocytochemical method employing E13 monoclonal anti- bodies specific for IE1 and 2 antigens (Seralab UK). PLoS ONE | www.plosone.org 7 March 2012 | Volume 7 | Issue 3 | e34119