Figure 6. Elevating NF-κB signaling inhibits HSV-2-induced KSHV replication.

(A). Transfection of IKK₂EE promoted the binding activity of P65 of NF-κB pathway in HSV-2-infected BCBL-1 cells. BCBL-1 cells were infected by HSV-2 or Mock for 1 hour, and then were transfected by IKK₂EE or MIGRI for 12, 24 and 48 hours. Cells were collected and nuclear proteins were extracted for ELISA-based NF-κB activity assay. Excess competitor oligonucleotides were coincubated with the nuclear protein of MIGRI-transfected and HSV-2-infected BCBL-1 cells for competition assays (HSV-2+MIGRI+Comp.). Results presented were the statistic of three independent experiments performed in triplicate. * p<0.05 and ** p<0.01 for Student's t-test versus Mock+MIGRI group, respectively; ^## p<0.01 and ^& p<0.01 for Student's t-test versus HSV-2+MIGRI group, respectively. (B). Transfection of IKK₂EE promoted HSV-2-induced activity of NF-κB in BCBL-1 cells. BCBL-1 cells were infected by HSV-2 or Mock for 1 hour, and then were co-transfected with the plasmid IKK₂EE and an NF-κB-dependent firefly luciferase reporter construct (0.4 µg) together with a Renilla luciferase construct pRL-TK (0.02 µg) for 12, 24 and 48 hours. Whole-cell extracts were prepared, and the firefly as well as the Renilla luciferase activity was measured. The experiment was performed three times in parallel, and the mean ± s.e.m is shown. ** p<0.01 and ^## p<0.01 for Student's t-test versus Mock+MIGRI and HSV-2+MIGRI groups, respectively. (C). Transfection of IKK₂EE inhibited ORF26 mRNA expression in HSV-2-infected BCBL-1 cells. BCBL-1 cells were infected by HSV-2 or Mock for 1 hour, and then were transfected by IKK₂EE or MIGRI for 12, 24 and 48 hours. Cells were collected and ORF26 mRNA expression in BCBL-1 cells was measured by RT-qPCR. Relative quantities of ORF26 expression represented on the y-axis. Results shown were from three independent experiments performed in triplicate. ** p<0.01 for Student's t-test versus Mock+MIGRI group; ^# p<0.05 and ^## p<0.01 for Student's t-test versus HSV-2+MIGRI group, respectively. (D). Transfection of IKK₂EE inhibited vIL-6 expression in HSV-2-infected BCBL-1 cells. BCBL-1 cells were infected by HSV-2 or Mock for 1 hour, and then were transfected by IKK₂EE or MIGRI for 12, 24 and 48 hours. Whole cell lysate were subjected to Western blot with the indicated antibody. (E). Real-time DNA-PCR analysis for the viral copy number of KSHV. BCBL-1 cells were infected by HSV-2 or Mock for 1 hour, and then were transfected by IKK₂EE or MIGRI. At 24 and 48 hours postinfection, supernatant virus was collected, concentrated, and used for real-time DNA-PCR analysis. Results shown were from three independent experiments performed in triplicate. ** p<0.01 and ^## p<0.01 for Student's t-test versus Mock+MIGRI and HSV-2+MIGRI groups, respectively.