FIG 5.
The KSHV latent gene K12 regulates the expression of NE genes in endothelial cells. (A) HMVEC-d were transduced with the control lentivirus vector pSIN or lentiviral vectors expressing ORFs 71, 72, 73, and K12. After 72 h posttransduction, cell lysates were Western blotted for ADM2, HRH1, NSE, PGP9.5, and SSTR1. β-Actin was used as a loading control. (B) qRT-PCR 72 h after transduction of HMVEC-d with lentiviral vectors showing the mRNA expression levels of latent ORFs 71, 72, 73, and K12. The comparative CT method was used to determine the expression levels of latent genes. (C) Knockdown efficiencies of K12 siRNAs. KSHV latently infected endothelial L1T2 cells were transfected with the siRNA negative control, K12 siRNA1, K12 siRNA2, and a mix of K12 siRNA1 and -2 for 72 h, and the cell lysates were Western blotted with anti-K12 antibodies. Percent inhibition indicates the inhibition of K12 expression in K12 siRNA-treated cells compared to control siRNA-treated cells. (D) K12 siRNA inhibits the expression of NE genes in KSHV latently infected L1T2 endothelial cells. NE protein levels in cells transfected with control siRNA, K12 siRNA1, K12 siRNA2, and a mix of siRNA1 and -2 for 72 h were measured by Western blotting. β-Actin was used as a loading control. Percent inhibition indicates the inhibition of NE gene expression in K12 siRNA-treated cells over that of control siRNA-treated cells.