Fig 4.

Expression of wild-type full-length (FL) ORF50 and truncated DN ORF50ΔSTAD after treatment with inducers of ORF50 and ORF50ΔSTAD expression and inducers of apoptosis. ORF50ΔSTAD BCBL-1 cells were treated with TPA to induce ORF50 expression and viral replication via the normal pathway, with a low concentration of DOX (0.1 μg/ml) to induce ORF50ΔSTAD expression, with DCPE to induce apoptosis, and with various combinations of the agents. (A) Expression of full-length wild-type ORF50 and ORF50ΔSTAD RNA, measured by real-time RT-PCR assays. (B) Expression of ORF50 and ORF50ΔSTAD proteins, assessed using immunoblots employing an antipeptide antibody made against an amino-terminal ORF50 peptide. TPA induces expression of FL ORF50 RNA and protein. DOX induces expression of ORF50ΔSTAD RNA and protein. When ORF50ΔSTAD is present, the expression of FL ORF50 is blocked at both the RNA and protein level. The immunoblot in panel B was deliberately overexposed to show that there is a very small amount of residual FL ORF50 expression in the cells prior to induction, slightly visible in the untreated and DCPE-only lanes. BCBL-1 cells spontaneously experience a small amount of lytic KSHV replication even without treatment with an inducing agent, which is suppressed when ORF50ΔSTAD expression is induced with DOX. (Bottom) The blot was reprobed with an antiactin monoclonal antibody (the antiactin-probed blot is a much shorter exposure).