Fig. 3.

Early increase in TXNIP and activation of the NLRP3 inflammasome precedes expression of the replication switch. (A and B) HH514-16 cells were exposed to lytic triggers NaB and 5-AZA-2′-deoxycytidine (versus dimethyl sulfoxide [DMSO] control) and harvested at indicated time points for immunoblotting with (A) indicated antibodies or (B) qRT-PCR. Numbers in A represent ratios between indicated proteins. Experiments were performed at least three times, and data in B represent averages of three independent experiments; error bars, SEM; *P ≤ 0.05. (C) HH514-16 cells were treated with NaB (versus untreated control) for 6 h, cell lysates were immunoprecipitated using indicated antibodies (IP), and bound proteins were detected by immunoblotting with indicated antibodies (IB). Input lysates are shown on the left. (D–F) CD19⁺ B cells from the blood of a patient with PTLD were analyzed by flow cytometry for EBNA2 and size to demarcate EBNA2⁺ “blast-like” large cells for further analysis; number indicates percent EBNA2⁺ cells (D). EBV-infected B blast-like cells were analyzed by flow cytometry for TXNIP (E) or cleaved caspase-1 (CASP1 p20; F) expression (Left dot plots), and percent TXNIP^hi versus percent TXNIP^lo cells (E) or percent CASP1 p20^hi versus percent CASP1 p20^lo cells (F) expressing the lytic switch protein ZEBRA are shown (Right dot plots). Data from a representative of 3 patients is shown.