Analysis of HHV-8–induced MCP-1 transcriptional activation. (A) Schematic representation of human MCP-1 reporter constructs.35 The proximal promoter region and distal enhancer region are indicated by open and closed boxes, respectively. The position of NF-κB sites (A1 and A2) in the enhancer region is indicated by open diamonds (⋄). Mutated NF-κB sites are indicated by filled diamonds (♦). (B) HUVECs were cotransfected with the pRL-SV40 vector together with one of the following plasmids: pGLM-PRM, pGLM-ENH, pGLM-MA1, pGLM-MA2, and pGLM-MA1A2, described in panel A. After 12 hours transfected cells were mock infected (control), infected with HHV-8, or treated with TPA (20 ng/mL). Dual luciferase activity was determined 30 hours after infection. The data, expressed as relative luciferase activity, represent the mean ± SD of quadruplicate samples from 2 independent experiments. (C) HUVEC monolayers were triple-transfected with the pRL-SV40 vector and the pGLM-PRM or pGLM-ENH reporter constructs together with “empty” (−) or IκBα-AA (+) vectors. After 12 hours, transfected cells were mock infected (− HHV-8) or infected with HHV-8 (+ HHV-8). Luciferase activities were determined 30 hours after infection as described in panel B. The data, expressed as relative luciferase activity, represent the mean ± SD of quadruplicate samples from 2 independent experiments.
