Expression of P-selectin on HUVECs is regulated by hypoxia and NO bioavailability and its role in sRBC adhesion. (A) Effects of hypoxia and the eNOS inhibitor l-NAME on P-selectin expression on HUVECs. P-selectin expression on the cell surface of HUVECs was evaluated by immunofluorescence microscopy. Cells were treated with room air (Fio₂ = 21%) or hypoxia of 12% O₂ for 1 hour with or without l-NAME (100 μM). Images were taken at ×400 (upper panel). MFIs were calculated by scanning fluorescence images with ImageJ v1.43 (NIH). Results with MFI for each experiment are shown in the lower panel. Values were mean ± SEM from 3 to 4 images taken for each treatment with at least 5 cells analyzed per image. (B) Analysis of P-selectin expression in HUVECs by western blotting. Cells were treated as described previously, and the cell membrane fractions were isolated and subjected to western blot analysis. Note that the intensity of a slower-mobility protein band at 140 kDa is increased in response to hypoxia and l-NAME treatment. (C) Effect of blocking antibodies on sRBC adhesion in eNOS-deficient mice under hypoxia. eNOS-deficient mice were infused with antibody against P-selectin, VCAM-1, and E-selectin or αvβ3-integrin blocking peptide before intravital experiments, and the adhesion scores of sRBCs were measured. Values were mean ± SEM from 5 to 7 mice in each group. P values are shown at the top of the figure.