HHV-8 induces arterial marker expression in Kaposi sarcoma cells. (A) Western blot for ephrin B2 on various cell lysates. SLK-vGPCR is a stable clone of SLK expressing the HHV-8 vGPCR, and SLK-pCEFL is control stable clone transfected with empty expression vector. SLK cells transfected with LANA or LANAΔ440 are SLK-LANA and SLK-Δ440, respectively. Quantity of protein loading and transfer was determined by reprobing the membranes with β-actin monoclonal antibody. (B) RT-PCR of stable cell lines shown in panel A, demonstrating expression of the relevant viral mRNAs. Housekeeping mRNA to demonstrate equivalent input mRNA and loading was β-actin or glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Specific bands of the expected sizes (Table 1) were obtained with all gene-specific primer pairs. (C) Transient transfection of KS-SLK cells with expression vector pCEFL-KSHV-GPCR resulted in the expression of ephrin B2 as shown by immunofluorescence staining with FITC (green), whereas the control vector pCEFL had no effect. KS-SLK cells (0.8 × 105/well) were transfected with 0.8 μg DNA using Lipofectamine 2000. Twenty-four hours later cells were fixed and stained with ephrin B2 polyclonal antibody and FITC-conjugated secondary antibody as described in “Patients, materials, and methods.” (D) Transient transfection of HUVECs with vGPCR induces transcription from ephrin B2 luciferase constructs. HUVECs (8 × 103) in 24-well plates were transfected using Superfect with 0.8 μg/well ephrin B2 promoter constructs containing sequences from -2941 to -11 with respect to the translation start site, or 2 5′ deletions as indicated, together with 80 ng/well pCEFL or pvGPCR-CEFL. Luciferase was determined 48 hours after transfection. pGL3Basic is promoterless luciferase control vector. Luciferase was normalized to protein since GPCR induced expression of the cotransfected β-galactosidase. Graphed is induction ratio (vGPCR/pCEFL) of mean ± SEM of 6 replicates. Shown is 1 of 3 similar experiments.
