CoPPIX induces DAF expression on vascular ECs. (A) HUVECs were treated for 24 hours with CoPPIX (up to 100 μM), and HO-1 expression was detected by immunoblotting. (B). HUVECs were treated for up to 24 hours with CoPPIX (50 μM). Total RNA was isolated; Northern blots prepared and probed for DAF mRNA. The fold change in DAF mRNA was calculated after densitometric scanning, normalized with respect to the ethidium bromide–stained 28S band. (C,D) HUVECs were cultured for 24 hours in the presence of CoPPIX or vehicle alone, and DAF expression was measured by (C) flow cytometry with results expressed as percentage increase in RFI above control ECs, treated with plain culture medium alone (mean ± SEM; n = 3 experiments) and (D) by immunoblotting. (E) HUVECs were infected with a HO-1 recombinant adenovirus (Adv–HO-1) at an MOI of up to 200 virus particles per cell or an empty vector control adenovirus (Ad0; MOI 200). ECs were lysed 24 hours after infection, and expression of HO-1, DAF, and α-tubulin was analyzed by immunoblotting. Relative levels of DAF protein expression were quantified using image analysis densitometry and expressed as the fold change above that in Ad0-infected ECs. (F) HUVECs were infected with Adv–HO-1 or Ad0 (MOI 200), and DAF expression was measured by flow cytometry 24 hours after infection, with results expressed as percentage increase in RFI above noninfected control ECs (mean ± SEM; n = 3 experiments). (G) HUVECs were treated for 8 hours with hemin (0.2 μM) in the presence or absence of ZnPPIX (15 μM) or vehicle alone (UT). DAF mRNA levels were quantified by real-time PCR, and data are mean plus or minus SEM (n =2 experiments) relative to untreated ECs. (H) HUVECs were left untreated (UT) or transfected with control siRNA (Ctrl) or HO-1 siRNA before treatment with hemin (Hem; 0.2 μM), CoPP (50 μM), or vehicle and DAF mRNA quantified by real-time PCR as above. *P < .05; **P < .01.