Figure 3: KSHV miRNAs inhibit IL6-dependent STAT3 activation and inhibit STAT3-dependent reporter activation.
From: Viral microRNAs Target a Gene Network, Inhibit STAT Activation, and Suppress Interferon Responses
(A) HUVECs were transfected with KSHV miRNAs (miR-K6-5, miR-K8 or miR-K9*) or control miRNAs (Neg). At 48 hpt, transfected cells were mock-treated or treated with IL6 for 20 minutes. Equal amounts of protein were probed for pY705-STAT3 and total STAT3 (top) and pS727-STAT3 and PKCδ (bottom). Relative levels of various proteins were normalized to actin, compared to the control miRNA (Neg), and plotted on the right (n = 4). (B) HUVECs were transfected with miR-K9 (that targets IRAK1) or siIRAK1 along with appropriate negative control RNAs. Lysates were probed for pY705-STAT3, pS727-STAT3, total STAT3 and IRAK1. Relative levels of various proteins were normalized to actin and plotted on the right (n = 4). (C) The changes in the levels of pY705-STAT3 relative to total STAT3 were measured from 3 A and 3B and plotted (n = 3). (D) 293 cells were reverse-transfected with the 4xM67 pTATA-Luc, pRNL-TK control and KSHV miRNAs or siRNAs. Cells were left untreated or treated with IL6 for eight hours and relative luciferase levels were measured as described in the Methods (n = 3). Full-length blots are in Figure S2.
