Reduction of PEL cell proliferation and induction of caspase activity by transient knock-down of vIRF-3 expression. JSC-1 cells were transfected with synthetic RNA oligonucleotides directed against vIRF-3 (panel A, si463; panel B, si976), nonsense siRNA (siN), or mock-transfected (mock) and cultured for 2 days. The KSVH⁻ cIRF-4 expression–positive myeloma cell line INA-6 was included as a control (C). At 2 days after transfection, two-thirds of the cells were harvested for both analysis of vIRF-3 expression by Western blot and enzymatic assay of caspase-3/7 activity. ³H-thymidine was added to the remaining cells, and proliferation was measured via ³H incorporation into DNA 24 hours later. Transfection of synthetic siRNAs resulted in 47% (panel A top) or 40% (panel B top) knock-down of vIRF-3 protein by si463 or si976, respectively. This was associated with a reduction of ³H-thymidine incorporation into DNA by 37% (panel A middle) or 28% (panel B middle), respectively. Reduced ³H-thymidine incorporation was accompanied by a pronounced increase of caspase-3/7 activity (panels A,B bottom). Expression of both cIRF-3 and cIRF-4 was not altered by any of the siRNAs in INA-6 cells (C). Cell proliferation and caspase-3/7 activity remained unchanged in these KSHV⁻ cells, whereas apoptosis could be effectively induced by etoposide treatment (etop.).