HO-1 expression following cell exposure to peroxide-treated and untreated Hb-Hp. (A) LightCycler quantification of HO-1 mRNA expression induced by oxidized and nonoxidized Hb and Hb-Hp in CD163-HEK cells after 8-hour incubation. Data were normalized for GAPDH expression and represent mean plus or minus SD from 3 independent experiments (● = nonoxidized; ○ = H2O2 treated). (B) HO-1 Western blot of CD163-HEK cell lysates after 12-hour incubation with oxidized (ox) or nonoxidized Hb/Hb-Hp. (c = nontreated cells.) (C) Subcellular localization of Hb 30 minutes after endocytosis of oxidized Hb-Hp (1-1) in CD163-HEK cells. Cells were stained with anti-Hb + Alexa 594 secondary antibody (red) and anti–LAMP-1 + Alexa 488 secondary antibody (green); nuclei were stained with DAPI (blue). The insert in the merged image demonstrates extensive colocalization of Hb with LAMP-1–positive lysosomes (colocalization appears yellow). Images were acquired with a Leica SP2-AOBS-UV confocal laser-scanning microscopy system (objective 40×/1.25, oil).
