Up-regulation of HO-1 in KSHV-infected DMVECs. (A) Age- and passage-matched uninfected and KSHV-infected DMVECs were harvested and lysed with 3 freeze/thaw cycles. Clarified supernatants from whole-cell extracts were resolved on SDS-PAGE, transferred to nitrocellulose membranes, and probed with a monoclonal antibody (mAb) raised against the 32-kDa protein HO-1. HO-1 expression was increased in KSHV-infected extracts (lane K) relative to uninfected extract (lane M). Treatment of KSHV-infected cells with the heme analogs CuMP and CrMP (lanes Cu and Cr, respectively) did not significantly alter HO-1 expression. SAPK/JNK was used as a loading control. (B) Monolayers of KSHV-infected DMVECs were fixed with 2% paraformaldehyde, permeabilized with 0.05% Triton X, and stained with an anti-HO-1 mAb and an anti-ORF 73 polyclonal antibody followed by FITC-labeled goat antimouse and Texas Red-labeled goat antirabbit secondary antibodies. HO-1 staining (green) was found exclusively in cells expressing ORF 73 (red), but not in uninfected neighboring cells, which are visible in the phase image in the right panel. (C) The recombinant EGFP-expressing clone rKSHV.152 was used to infect primary DMVECs at a low multiplicity of infection. Monolayers were fixed and permeabilized as described in B and stained for HO-1 (red). EGFP-KSHV-positive, spindle-shaped DMVECs stained positive for HO-1 (red), whereas uninfected EGFP-negative cells, visible in the phase image in right panel, did not. Original magnification, × 20.