HEK293 cells were infected as per earlier procedures (25) . Figure 6 . RDS reduces the Egr-1/ORF50 association in vivo. (A) RDS lowers Egr-1 and KSHV RTA expression. KSHV-infected BCBL-1 cells were synchronized in S phase and untreated or treated using 20 ng/ml of TPA for 2 h. Each group of cells was left untreated or was further treated using filtered or unfiltered RDS containing 100 mM of resveratrol. The cells were incubated at 37uC for 6 h and lysed. The lysates were resolved on a 10% SDS-PAGE, transferred to a PVDF membrane, and Western blotting was performed using specific antibodies. (B) RDS reduces the number of KSHVinfected cells undergoing reactivation in HEK293 cells. Mock-infected, KSHV-infected, and KSHV-infected cells in the presence of RDS containing 100 mM resveratrol were stained using monoclonal mouse anti-Egr-1 IgGs and rabbit peptide antibodies targeting KSHV RTA and examined under a fluorescent microscope (magnification 6100). (C) Overexpression of Egr-1 is unable to overcome RDS-mediated inhibition of virus reactivation. BCBL-1 cells were transiently transfected using pcDNA3.1(+) or egr-1/pcDNA3.1(+) and subsequently treated with unfiltered RDS containing 100 mM of Resveratrol for 6 h. RNA was extracted, cDNA was synthesized, and egr-1 and KSHV ORF50 were analyzed by qRT-PCR. Each point denotes the average6S.D. (error bars) of three experiments. (D) RDS lowers egr-1 and KSHV ORF50 transcriptional activity. BCBL-1 cells were synchronized in S phase, treated with 20 ng/ml of TPA, and treated using filtered or unfiltered RDS containing 100 mM of resveratrol as before. RNA was extracted, cDNA was synthesized, and egr-1 and KSHV ORF50 were analyzed by qRT-PCR. Each point denotes the average6S.D. (error bars) of three experiments. Columns with different alphabets are statistically significant (P,0.05) by LSD. (E) RDS inhibits the ability of Egr-1 to bind KSHV ORF50 in vivo. BCBL-1 cells were synchronized and treated with TPA as before. The cells were further treated using unfiltered RDS containing 100 mM of resveratrol and incubated for 6 h. ChIP assays were performed using 2 mg of specific Egr-1 Abs. Semi-quantitative PCR experiments were performed using samples from 1% input DNA and IP samples in order to determine the expression of ORF50P8. Respective cDNA at 10, 15, 20, 25, and 30 cycles were resolved on a 2% agarose gel. Specific antibodies to histone H3 and nonspecific IgGs were used to IP sample chromatin and served as positive and negative controls, respectively. doi:10.1371/journal.pone.0033364.g006