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. 2012 May 22;26(11):2343–2352. doi: 10.1038/leu.2012.108

Figure 2.

Figure 2

EBNA2 and miR-21 expression in EBV-infected and EBNA2-transfected B tumor cells. (a) U2932 (DLBCL), Bjab (B-cell lymphoma) and Oma 4 (BL) were infected with the recombinant Akata strain of EBV. EBNA2 expression in the resulting convertants is shown by immunoblotting, using PE2 monoclonal antibodies. β-Actin expression was verified as loading control. The lower panels show miR-21 expression by northern blotting. End-labeled LNA antisense oligonucleotide was used as a probe. As a control for comparable loading and RNA quality, U2 expression was analyzed. ‘*' indicates a P-value of <0.05 by one sided t-test. (b) miR-21 expression was verified in isogenic pairs of BL28 and BL41, infected with the wild-type B95-8 and EBNA2 lacking P3HR1 viral strains. The upper panels show EBNA2 expression as detected by immunoblotting. The lower panels show miR-21 expression by northern blotting. The histograms are mean (±s.d.; n=3) densitometric analysis of northern blot data on miR-21 expression. The fold changes are normalized values against the house keeping U2 RNA. (c) An expression vector for EBNA2 was electroporated into U2932, Oma 4 and BL41 cells. EBNA2 expressing clones were established from all three cell lines. The expression of EBNA2 was analyzed by immunoblotting with PE2 monoclonal antibodies. β-Actin expression is used as loading control. miR-21 expression as evaluated by northern blotting is shown in the lower panels. (d) Estradiol-inducible EBNA2 was transfected into Bjab (Bjab K3) and BL41 (BL41 K3) cells. Estradiol-treated and -untreated cells were tested for EBNA2 and miR-21 expression. The ER–EBNA2 fusion protein was of higher molecular weight, EBNA2 induction was accompanied by an increase in miR-21 expression. β-Actin expression is the protein loading control and U2 expression is RNA loading and quality control. B95-8 was used as positive control both for EBNA2 and miR-21. The mean (±s.d.; n=3) densitometric analysis of miR-21 northern blots is shown below. The fold changes are normalized values against the house keeping U2 RNA.