Original magnifications, × 10. (C) Quantification of results in B. *** P < 0.001 for Student's t-test versus Mock + pSRα group, whereas **P < 0.01 for Student's t-test versus HSV-1

+ pSRα group. 3.3. Both overexpression of PTEN and activation of GSK-3β pathway also inhibit HSV-1-induced KSHV reactivation From Figure 2, we observed that expression of PTEN (negative regulator of PI3K/AKT pathway) was low in HSV-1-infected BCBL-1 cells, therefore, we asked whether overexpression of PTEN could influence HSV-1-induced KSHV replication. To address this issue, the PTEN cDNA construct was transfected to the cells. Western blot analysis demonstrated that overexpression of PTEN not only decreased phosphorylated GDC-0068 nmr AKT and GSK-3β (data not shown), but also reduced HSV-1-induced KSHV Rta and vIL-6 proteins expression (Figure 5A). To further determine whether overexpression of PTEN could reduce the release of KSHV progeny virions induced by HSV-1, experiments were designed to detect the copy number of KSHV progeny virions. The results of real-time DNA-PCR demonstrated that the copy number of KSHV virions in the supernatant from PTEN-transfected and HSV-1 infected BCBL-1 cells was significantly decreased when compared

to those from pcDNA-transfected and HSV-1 infected BCBL-1 cells (Figure 5B). Figure 5 Overexpression of PTEN and activation of GSK-3β inhibit HSV-1-induced KSHV reactivation. (A) Western blot analysis was used to detect the expression of KSHV Rta, vIL-6 and the level of the transfected PTEN in PTEN or selleck chemicals llc control vector transfected and HSV-1 infected BCBL-1 cells as indicated. (B) Real-time DNA-PCR was used to detect the copy number of KSHV progeny virions in the supernatant of PTEN or control vector transfected and HSV-1 infected BCBL-1 cells as indicated. ** p < 0.01 and ## p < 0.01 for Student's t-test versus Mock + pcDNA and HSV-1 + pcDNA groups, respectively. (C) Western blot analysis was used to detect the expression of KSHV Rta, vIL-6 and the level of the transfected GSK-3β-S9A

in GSK-3β-S9A or control vector transfected and HSV-1 infected BCBL-1 cells as indicated. Because HSV-1 infection of BCBL-1 cells increased phosphorylated GSK-3β (Figure 2) and transfection of PI3K-DN decreased Docetaxel mw HSV-1-induced phosphorylation of GSK-3β (Figure 3C), we reasoned that inactivated GSK-3β might www.selleckchem.com/products/BIBW2992.html promote HSV-1-induced KSHV replication. To test this hypothesis, the GSK-3β mutant plasmid GSK-3β-S9A, which exhibits constitutively active GSK-3β, was transfected to BCBL-1 cells. As expected, the expression of KSHV Rta and vIL-6 proteins in GSK-3β-S9A-transfected and HSV-1 infected BCBL-1 cells was markedly reduced compared to pcDNA-transfected and HSV-1 infected BCBL-1 cells (Figure 5C). Taken together, these data suggest that PTEN/PI3K/AKT/GSK-3β pathway may play an important role in HSV-1-induced KSHV reactivation. 3.4.