Original magnifications, × 10 (C) Quantification of results in B

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 AZD6244 purchase 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 Opaganib mw 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 STK38 HSV-1-induced phosphorylation of GSK-3β (Figure 3C), we reasoned that inactivated GSK-3β might 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.

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