Bar chart summary of microarray expression profiling comparing genes regulated by ER-EBNA2 type 1 and type 2 stably transfected in AK31 BL cells. Cells were pretreated for 1 h with protein synthesis inhibitors and then with or without estrogen for 4 h in the presence of protein synthesis inhibitors. Total cell RNA was extracted and used for expression profiling on Agilent 44K microarrays (as described in Materials and Methods section). The experiment was performed in duplicate, and the resulting RNAs were hybridized pairwise (estrogen-treated versus control) on individual microarrays. For each condition the values were averaged and a standard error was calculated. Each bar represents the ratio of the mean of values with estrogen over the mean of the values without estrogen (fold induction). The inset shows Western blotting using PE2 antibody of ER-EBNA2 levels in the AK31:EBNA1/ER-EBNA2 cells with or without 4 h of treatment with estrogen.

(A) Left panels, estrogen-starved EREB2.5 cells (E2.5) were treated with estrogen for 4 h, and RNA was extracted and analyzed by RT-PCR for CXCR7 mRNA using the indicated primer combinations (right panels) to detect alternately spliced forms. Middle panels, estrogen-starved EREB2.5 cells (E2.5) or AK31:EBNA1/ER-EBNA2 type 1 or type 2 cells (AK31) were pretreated with protein synthesis inhibitors for 1 hour (AK31) or 2 h (E2.5), and the cells were then treated with or without estrogen for 4 h in the presence of protein synthesis inhibitors (as in the microarray experiment in Fig. 2). RNA was extracted and analyzed by RT-PCR for CXCR7 mRNA using the indicated primer combinations (right panels). Lanes M, size markers (100-bp ladder). (B) RT-PCR assay for CXCR7 or ADAMDEC1 mRNA in EREB2.5 cells at 7 or 10 days after transfection with EBNA2 T1 or T2 vector and withdrawal of estrogen as for Fig. 1. CXCR7 primers were those shown in the upper panels of panel A. GAPDH mRNA was also assayed to ensure equal input RNA in the samples. (C) RT-PCR assay for CXCR7 mRNA in a panel of LCLs with the primers used in upper panels of panel A. GAPDH mRNA was also assayed as a control.

(A) Type 1 and type 2 EBNA2 both repress MYC expression and drive apoptosis in AK31 BL cells. AK31:EBNA1/ER-EBNA2 T1 and T2 stable cells were induced with 5 μM β-estradiol for 0 to 72 h. Extracts from cells were probed with indicated antibodies. (B) Analysis of EBNA2 T1- and T2-driven apoptosis in AK31 cells. AK31:EBNA1/ER-EBNA2 T1 and T2 stable cells induced with 5 μM β-estradiol for 0 to 72 h were ethanol fixed and stained with propidium iodide prior to analysis of DNA content by flow cytometry. Upper panels, DNA content analysis of AK31:EBNA1/ER-EBNA2 cells following ER-EBNA2 T1 activation, demonstrating a transient increase in G₀/G₁ populations followed by apoptosis at later time points. Numbers inserted into the histogram profiles denote the percent subdiploid, G₀/G₁, S, and G₂/M populations, respectively. Lower panels, bar chart comparing subdiploid and G₀/G₁ DNA contents for both T1 and T2 ER-EBNA2 stable populations. (C) Type 1 and type 2 EBNA2 drive mitochondrial/intrinsic apoptosis in AK31 cells. AK31:EBNA1/ER-EBNA2 T1 and T2 stable cells were induced with 5 μM β-estradiol for 0 to 72 h and analyzed for apoptosis. The JC-1 assay was performed at each time point. The upper panel shows example profiles of carbonyl cyanide m-chlorophenylhydrazone-treated positive control at 0 h and 72 h with or without activation of T1 ER-EBNA2 stable cells. The bar chart (lower panel) shows a comparison between EBNA2 T1/T2-driven intrinsic apoptosis or the percentage of normal (N) cells versus apoptotic (Ap) cells in both cell populations. (D) DNA laddering analysis was also performed on AK31:EBNA1/ER-EBNA2 T1 or T2 cells induced with 5 μM β-estradiol for 0 to 72 h.

(A) EREB2.5 cells which had been growing normally in estrogen were washed to remove estrogen and transfected with EBNA2 T1 or T2 vector expressing EBNA2 type 1 or type 2 protein. Cells were cultured in the absence of estrogen and pulse-labeled at the times indicated with [³H]thymidine. (B) Phase-contrast images of cells from panel A at 7 days after transfection of EBNA2 plasmid, showing proliferating cells only with type 1 EBNA2. The lower panel shows a Western blot for EBNA2 in three LCLs after 3 months of outgrowth. (C) Western blots of extracts from the same transfections assayed in panel A at 3 or 4 days after transfection as indicated for EBNA2, LMP1, IRF4, RUNX3, and actin as a loading control. (D) Comparison of detection of in vitro-translated type 1 and type 2 EBNA2 by Western blotting with PE2 antibody. Relative detection by Western blotting (WB) is similar to that by [³⁵S]Met labeling during the in vitro translation.

(A) ELISA detection of IL-1β. AK31:EBNA1/ER-EBNA2 type 1 or type 2 cells were cultured with or without estrogen for 24 h. The medium and cell extracts (IC) were assayed for IL-1β by ELISA. (B) Cells were treated as for panel A and analyzed by Western blotting for IL-1β. (C) Cells were treated as for panel A and analyzed by RT-PCR for ADAMDEC1 and GAPDH as a positive control. Error bars indicate standard errors.

(A) Plasmids expressing shRNA for CXCR7 or p53 as a negative control or the control empty vector (V) were transfected into EREB2.5 cells, and the transfected cells were selected with puromycin. Live cell counts (trypan blue exclusion) at the indicated times after transfection are shown. CXCR7 RNA was assayed by RT-PCR at 7 days after transfection using the exon 2 primer combination shown in the upper panels of panel A. (B) As for panel A but with LCL3 cells (an LCL infected with B95-8 EBV). Here the CXCR7 RNA was assayed by RT-PCR at 6 days after transfection. (C) To show that the CXCR7 shRNA plasmid was not nonspecifically toxic to cells and that it did not prevent function of the puromycin resistance gene, the shRNA plasmids used for panels A and B were transfected into 293 cells and selected with puromycin. The control (Ctrl) sample received a plasmid lacking the puromycin resistance gene. Live cell counts at 8 days after transfection are shown. Error bars indicate standard errors.

Daudi BL cells stably transfected with ER-EBNA2 type 1 or type 2 were cultured with or without estrogen for the indicated times, and protein extracts of the cells were analyzed by Western blotting for EBNA2 (PE2 antibody), LMP1 (CS1-4 antibody), or actin as a loading control.