Induction of MCP-1 expression by HHV-8 infection in endothelial cells. (A) HUVECs were mock infected (control) or infected with HHV-8 (HHV-8). At 3 and 24 hours after infection total RNA was extracted and analyzed by RT-PCR for the expression of IL-8, MCP-1, RANTES, and TNF-α. β-Actin levels are shown as control. (B) In the same experiment culture supernatants were collected at 3 and 24 hours after infection and analyzed for the release of the indicated proinflammatory mediators by standard quantitative ELISAs. Levels of chemokines are expressed as fold induction of the levels detected in mock-infected control cells. Bars represent the mean ± SD of triplicate samples. (C) Mock-infected and HHV-8–infected HUVECs were harvested at the indicated times, and MCP-1 and β-actin mRNA levels were analyzed by TaqMan rtPCR. MCP-1 mRNA levels normalized for β-actin levels in the same samples are expressed as fold induction of the levels detected in mock-infected cells at the same time points. Bars represent the mean ± SD of triplicate samples. (D) In the same experiment MCP-1 levels in culture supernatants from mock-infected (−) and HHV-8–infected (+) cells were determined by ELISA. The results represent the mean ± SD of triplicate samples. (E) Cycloheximide and monensin (Sigma-Aldrich) were used to inhibit protein synthesis and secretion, respectively. Mock-infected (empty symbols) or HHV-8–infected (filled symbols) HUVECs were treated with 5 μM cycloheximide (□, ▪), 5 μM monensin (▴, ▵), or control diluent (○, •) 8 hours after infection (indicated by arrow). MCP-1 levels in culture supernatants were determined 24 hours after infection by ELISA. Bars represent the mean ± SD of triplicate samples. All experiments were performed 3 times with similar results.