Characterization of lytic and latent phases during long term HHV8 infection of HUVEC cells. HUVEC cells were infected with HHV8, concentrated at a multiplicity of at least 10-20 genomes per cell in a M200 medium containing 2 μg/ml of polybrene for 2 h at 37°C. Panel A: On days 3, 14 and 24 post infection, 1.0 × 106 cells were harvested and RNA-extracted for detection of lytic (orf26 and orf50) and latent (orf73) viral genes by RT-PCR. HHV8-infected BC3 cells were used as a positive control. The housekeeping β-actin gene was used as a control (for details see Methods). Panel B: At the indicated times (3, 14 and 24 days post infection) sub-confluent cells were harvested and processed as required for western blotting (for details see Methods). Panel C: 24 h before the indicated times (3, 14 and 24 days post infection), cells were seeded at a density of 2.0 × 105 in 35 mm glass-bottomed dishes and cultured at 37°C in a 5% CO2 incubator in a growth medium. Thereafter, cells were washed and fixed for the immunofluorescence detection of lytic (K8.1) and latent (LANA) viral-antigens (green) as reported in Methods. The bar in the figure is 30 μm.