HHV-8 induces arterial marker expression in venous endothelial cells. (A) RT-PCR of HUVECs, 2 HHV-8-infected cultures (HUVEC/BC-1 and HUVEC/BC-3), and BC-1 for ephrin B2 and EphB4. Ephrin B2 product (200 bp) is seen in HUVEC/BC-1 and HUVEC/BC-3, and EphB4 product (400 bp) is seen in HUVECs and BC-1. No template lane had exactly the same reaction components and conditions, except template cDNA was omitted. This is included to show the specificity of the amplified products. β-Actin RT-PCR is also shown as a control for amount and integrity of input RNA. (B) Western blot of protein extracts of BC-1 and BC-3 HHV-8-positive cell lines. Membranes were sequentially probed with ephrin B2, EphB4, and β-actin monoclonal antibodies. Specific bands were detected at 120 kDa for EphB4, 37 kDa for ephrin B2, and 40 kDa for β-actin. (C) Immunofluorescence of cultures of HUVECs and HUVEC/BC-1 for artery/vein markers and viral proteins. Cultures were grown on chamber slides and processed for immunofluorescence detection of ephrin B2 (i, v, ix), EphB4 (xiii, xvii, xxi), CD148 (x, xxii), and the HHV-8 proteins LANA1 (ii, vi, xiii) or ORF59 (xviii) as described in “Patients, materials, and methods.” Yellow color in the merged images of the same field demonstrate coexpression of ephrin B2 and LANA or ephrin B2 and CD148. The positions of viable cells were revealed by nuclear staining with DAPI (blue) in the third column (iii, vii, xi, xv, xix, xxiii). Photomicrographs are of representative fields.