A. CD20 expression enhances IFN promoter reporter activity.

<p>293T cells were transfected with IFN-β promoter reporter constructs and expression plasmid of CD20 (0.1, and 0.2 mg). After the overnight incubation, Sendai virus (20 HA units/ml) was added and the luciferase and β-galactosidase activities were measured 24 hours later. The relative fold of activation was calculated as a ratio of luciferase over β-galactosidase activities. Standard deviations are as shown. <b>B</b>. <b>Rituximab enhances VSV-induced IFN-α production</b>. IB4 cells were treated with VSV (M.O.I=1) and Rituximab. 24 hours later, medium was collected for ELISA for IFN-α (multiple subtypes) measurements. Samples were measured in duplications, and the average of duplications is as shown.</p

Dose and time-dependent enhancement of the IFN-production by Rituximab.

<p>A. Different amounts of Rituximab and constant Sendai virus (200 HA units/ml) were used to treat IB-4 cells simultaneously. The RNA was isolated after 6 hours and RPA was used for detection of IFN productions. B. the IB4 cells were treated with 10 mg/ml Rituximab with indicated time, Sendai was then used to infect the cells. The RNA was isolated after 6 hours and RPA was used for detection of IFN productions. Specific protections of IFN-β and GAPDH RNAs are indicated.</p

IRF-7C has oncogenic potential.

<p>(A) IRF-7C causes anchorage-independent growth of NIH 3T3 cells. A soft-agar assay was used for an IRF-7C stably expressing cell line established in NIH 3T3 cells. The numbers are averages from three independent experiments and standard deviations are also shown. (B) IRF-7C partially restores the growth property of IRF-4-knockdown cells. Knockdown of IRF-4 inhibits the growth of EBV-transformed cells. An EBV-transformed cell line (IB4) was transfected with shLuc, shIRF4, shIRF4 plus IRF-7C, or shIRF4 plus IRF7C-K92E by use of an Amaxa Nucleofector device respectively. One day after transfection, live cells were isolated and seeded. At the indicated days after transfection, surviving cells were enumerated by trypan blue exclusion. Each point represents the number of live cells (mean ± standard deviation) from three different counts. The difference between shIRF4, and shIRF4+IRF-7C is statistically significant (p = 0.0071). One representative of three independent experiments is shown.</p

Expression of IRF-7C RNA is associated with type III latency.

<p>(A) Schematic diagram of IRF-7C-specific RPA probe. The bar represents ORF. The IRF-7C-specific RPA probe encompasses the splicing junction. The IRF-7-specific probe region for all splicing variants is also shown. The drawing is not on scale. (B) IRF-7C RNA is highly expressed in cells with EBV type III latency. Total RNAs from the indicated cells lines were used for RPA with IRF-7C and GAPDH-specific probes. The images in the same box indicate that they are derived from the same gels. The identities of RNAs are shown.</p

K-RTA inhibits EBV lytic gene expression.

<p>A. K-RTA inhibits the EBV lytic gene expression in Akata cells. Akata (EBV+/KSHV-) cells were transfected with K-RTA or vector plasmid (5 µg DNA). EBV lytic replication was induced by anti-human-IgGs a day later (0, 1, 5, 10 µg/ml). Cell lysates were separated on SDS-PAGE, transferred, and used for western blot analysis. The identity of proteins is as shown. B. K-RTA inhibits the EBV lytic gene expression in AGS-BX11g cells. AGS-BX11g (EBV+/KSHV−) cells were infected with recombinant adenovirus expressing K-RTA or GFP (10 pfu/cell). EBV lytic replication was induced by TPA a day later. Lysates were used for western blot analysis. The same membrane was stripped and reprobed with other antibodies. One representative from three experiments is shown. The identity of proteins is as shown.</p

IRF-7C inhibits IRF-7-mediated transactivation of IFN promoter.

<p>A. IRF-7C inhibits the IRF-7-mediated activation of IFN-β reporter construct. 293T cells were transfected with 0, 50, 100, and 200 ng of IRF-7C (lanes 1–4 respectively) plus IFN-β promoter reporter construct and β-gal expression plasmids. Lanes 5–8, based on the same conditions of lanes 1–4, IRF-7A plasmid (100 ng) was added and also co-transfected into 293T cells with other plasmids. (B) DNA-binding of IRF-7C is required to inhibit the activation of IFN-β promoter. 293T cells were transfected with pcDNA3, IRF-7A (100 ng) IRF-7A+IRF-7C (200 ng), and IRF-7A+IRF7C-K92E (200 ng) respectively (lanes 1–4), along with IFN-β promoter reporter construct and β-Gal expression plasmids. Luciferase and β-gal activities were measured at 24 h after transfection. The relative folds of activation of promoter constructs are shown with standard deviations. One representative of three independent experiments is shown.</p

IRF-7C is associated with EBV transformation of primary B lymphocytes in vitro.

<p>Top panel: IRF-7C is highly expressed in EBV-transformed cells. Primary B cells were isolated from fresh human blood by the use of CD19 antibody-conjugated magnetic beads. Lysates from primary B cells from two individuals (lanes 1 and 2) and EBV-transformed B cell lines (lanes 3–6) were separated by 12% SDS-PAGE. The expression levels of IRF-7C and GAPDH were determined by Western blotting. The same membrane was stripped and reprobed with GAPDH antibody. Bottom panel: relative levels of IRF-7C expression in EBV transformed cells. The relative levels of IRF-7C expression (IRF-7C/GAPDH) were obtained by measuring intensity of IRF-7C and GAPDH from Panel A using ImageJ 1.37v software (NIH).</p

LMP-1 stimulates the expression of IRF-7C.

<p>DG75 cells were transfected with pcDNA3, LMP-1, or LMP-DM expression plasmids. The transfected cells were isolated, and total RNAs were isolated and used for RPA with IRF-7C and GAPDH-specific probes. Yeast RNA was used as negative control. Specific protections of IRF-7C and GAPDH RNAs are indicated.</p

Initiation of KSHV lytic gene expression correlated with the reduction of EBV lytic gene expression.

<p>A. TPA induces either EBV or KSHV lytic replication. BC1 (EBV+/KSHV+) cells were treated with TPA at indicated dosages shown on the top. Cell lysates were used for western blot analyses a day later. The same membrane was stripped and reprobed with other antibodies. The identity of proteins is as shown. The relative levels of EA-D expression (EA-D/Tubulin) and EBV-Z (EBV-Z/Tubulin) were obtained by measuring intensity of EA-D, EBV-Z, and Tubulin using ImageJ 1.37v software (NIH), and are shown on the bottom panels. One representative from three independent experiments is shown. B. Kinetics of K-RTA expression in BC1 cells. BC1 cells were treated with TPA (20 ng/ml), or butyrate (3 mM), or both. Total RNA were isolated at indicated time post treatment. The expression of K-RTA and GAPDH RNA was monitored by RPA with K-RTA and GADPH probes simultaneously. Specific protections of K-RTA and GAPDH RNAs are indicated. The relative levels of K-RTA RNA expression (K-RTA/GAPDH) were obtained by measuring intensity of K-RTA and GAPDH using ImageJ 1.37v software (NIH), and are shown on the bottom. One representative from three independent experiments is shown.</p

K-RTA inhibits EBV-Z-mediated EBV lytic gene expression.

<p>A. K-RTA inhibits EBV-Z-mediated EBV lytic gene expression. EBV-Z expression plasmid (0, 0.1, and 0.2 µg) plus K-RTA (0.2 µg) were transfected into BRLF1KO (EBV+/KSHV−) cells in 6-well plate as shown on the top. Lysates were used for western blot analysis 24 hours later. The same membrane was stripped and reprobed with other antibodies. The identity of proteins is as shown. B. Dose-dependent inhibition of EBV-Z-mediated lytic gene expression by K-RTA. Fix amount of EBV-Z expression plasmid (0.1 µg) plus various amounts of K-RTA (0, 0.05, 0.1, 0.2, 0.4 µg) were transfected into BRLF1-KO (EBV+/KSHV−) cells in 6-well plate as shown on the top. Lysates were used for western blot analysis. Same cell lysates were used. C. K-RTA inhibits the synergistic activation of EA-D. EBV-Z expression plasmid (0.025 µg), E-RTA (0.1 µg), and K-RTA (0.2 µg) were transfected with different combinations into BRLF1-KO cells as shown on the top. The same cell lysates were used for western blot analysis. The identity of proteins is as shown.</p