Figure 3.

Up-regulation of EZH2 in KSHV-infected endothelial cells is mediated by the NF-κB pathway, and by viral latent genes vFLIP and LANA. A, Inhibition of the NF-κB pathway decreases the expression of EZH2 protein. Cells were either treated with Bay-11 at the indicated concentrations for 24 h (SLKp and BOEC cells at the upper and middle panels, respectively) or transduced with IκBαM for 48 h (SLKp cells at the lower panel), and examined for the expression of EZH2 protein. B, Inhibition of the NF-κB pathway decreases the mRNA expression and promoter reporter activity of EZH2 in SLKp cells. For mRNA detection, cells were treated with 2.5 µM Bay-11 for 16 h and examined for the expression of EZH2 mRNA by RT-qPCR using GAPDH mRNA for normalization (left panel). For reporter assay, cells transfected with EZH2 promoter firefly luciferase reporter construct for 24 h, split and treated with 2.5 µM Bay-11 at 38 h post-transfection for another 10 h were examined for luciferase activities (right panel). In both cases, the levels of untreated cells was set as “1”. C, vFLIP and LANA up-regulate the expression of EZH2 protein and mRNA in BOEC cells. Cells transduced with vFLIP (upper and middle panels), LANA (lower panel) or control lentivirus for 48 h with or without further treatment with 2 µM Bay-11 for an additional 24 h (vFLIP) were examined for the expression of EZH2 protein (vFLIP- and LANA-transduced cells) and mRNA (vFLIP-transduced cells). Expression of vFLIP and LANA was detected by measuring the expression of mRNA (vFLIP) and protein (LANA), respectively. The expression of GAPDH was used for normalization. D, Conditioned medium has no effect on EZH2 expression. Uninfected BOEC cells were treated with conditioned medium from either mock- or KSHV-infected BOEC cells for 24 h, and examined for the expression of EZH2 protein.