Ifferent mechanisms, the anthracycline DNA intercalator doxorubicin, the vinca alkaloid microtubule inhibitor vincristine, as well as the glucocorticoid prednisone. In these experiments (Fig. six), we assessed apoptosis flow cytometrically and in parallel evaluated herpesvirus replication by assaying for protected herpesvirus DNA inside the cell supernatants. We identified that doxorubicin, vincristine, and prednisone induced apoptosis in all the herpesvirus latently infected cell lines and induced herpesvirus replication in these cell lines in a caspase-3dependent fashion. These studies also confirmed, utilizing a diverse flow-cytometric approach than the method applied within the experiments shown in Fig. 1 to 4, that TPA induced viral replication with out inducing apoptosis, that DCPE induced both apoptosis and viral replication, and that the induction of viral replication by apoptosis essential caspase-3 activity.October 2013 Volume 87 Numberjvi.asm.orgEBVViral DNA (copies/l)2000 1500 1000 500Percent Dead Cells80 60 40 20TP Aat ed TP AedrentUHHV-6AViral DNA (copies/l)2000 1500 1000 500Percent Dead Cells80 60 40 20UntDCPE + [Caspase-3 Inhibitor (M)]reDCPE + [Caspase-3 Inhibitor (M)]atnt re at ed TP Aat ed TP AUHHV-6BViral DNA (copies/l)2000 1500 1000 500Percent Dead Cells80 60 40 20UntreDCPE + [Caspase-3 Inhibitor (M)]DCPE + [Caspase-3 Inhibitor (M)]edat ed TP ATP ArentUHHV-Percent Dead CellsViral DNA (copies/l)80 60 40 202500 2000 1500 1000 500TP AedrentUKSHV80 60 40 20Viral DNA (copies/l)Percent Dead CellsU4000 3000 2000 1000ntreDCPE + [Caspase-3 Inhibitor (M)]at ed TP AUntreDCPE + [Caspase-3 Inhibitor (M)]DCPE + [Caspase-3 Inhibitor (M)]atatDCPE + [Caspase-3 Inhibitor (M)]TP Aat ed TP AedrentFIG 4 The proapoptotic agent DCPE induces cell death and viral replication in cell lines latently infected with herpesviruses inside a caspase-3-dependent process.Cell lines latently infected with herpesviruses, BCBL-1 cells latently infected KSHV, LCLa cells latently infected with EBV, HSB2 cells latently infected with HHV-6A, Z29/SupT-1 cells latently infected with HHV-6B, and SupT-1/JI cells latently infected with HHV-7 were treated with TPA to induce viral replication by means of the conventional pathway as a optimistic control and with the proapoptotic agent DCPE to induce apoptosis. Aliquots of your cells treated using the apoptotic inducer DCPE had been also treated with many concentrations of a caspase-3 inhibitor. Cells had been stained with annexin V-FITC and propidium iodide (PI) and assayed for apoptosis making use of flow cytometry.82409-02-7 Chemscene Cell supernatants had been assayed for viral DNA replication making use of a viral DNA TaqMan assay.2,2′-Dibromo-1,1′-biphenyl Purity DCPE induced apoptosis, which was blocked by the caspase-3 inhibitor inside a dose-dependent manner.PMID:33622227 All of the herpesvirus latently infected cell lines induced into apoptosis by DCPE made significant amounts of virus, which was blocked in a dose-dependent manner by the caspase-3 inhibitor.jvi.asm.orgUUntreDCPE + [Caspase-3 Inhibitor (M)]DCPE + [Caspase-3 Inhibitor (M)]atJournal of VirologyApoptosis Activation of Herpesvirus Replication900 Untreated 800 TPA DCPEProtected Viral DNA (copies/uL)HHV-6AHHV-6BHHV-FIG 5 Production of infectious virions following induction of viral replication by the apoptotic inducer DCPE. Supernatants from the cell lines latently infectedwith HHV-6A, HHV-6B, or HHV-7, induced into replication with either the conventional inducer, TPA, or the inducer of apoptosis, DCPE, were added to uninfected Jurkat cells, and subsequently supernatants from.