Deregulation of intracellular iron in SCD PBMCs. (A) Increased ferritin expression in SCD PBMCs. PBMCs obtained from 3 SCD patients and 3 controls were cultured in media supplemented with PHA for 36 hours and then activated with IL-2 for 36 hours. Where indicated, control PBMCs were further treated with 100 μM hemin for 24 hours. Ferritin was measured in cell lysates by ELISA. The means ± SD are shown. (B) Reduced intracellular iron levels in SCD PBMCs. Labile intracellular iron pool was measured in PBMCs obtained from 3 SCD patients or 3 normal controls. Cells were treated with 0.1 μM calcein AM for 10 minutes at 37°C. After washing with PBS, cells were incubated at 37°C, and calcein fluorescence was measured in a Glomax Multidetection system at different time points. Fractional fluorescence (F − F₀)/F, which is inversely proportional to the amount of chelatable iron plotted on the y-axis. Paired Student t test was used to determine P value of the iron values at 30 minutes. (C-G) Elevated expression of TFR, HIF-1α, ferroportin, IKBα, and HO-1 in SCD PBMCs. Cell lysates from activated PBMCs obtained from SCD patients and normal controls were resolved on 4% to 12% Bis-Tris gel and probed with antibodies against TFR, HIF-1α, ferroportin (FPN), IKBα, and HO-1. The β-actin was used as loading control. Results were quantified using Image Quant Software. Bars represent independent experiments on 4 patients and 4 controls for HIF-1α and IKBα; 6 SCD patients and 6 controls for ferroportin; and 3 SCD patients and 3 controls for HO-1. The blots show 2 representative SCD and control samples. The means ± SD are shown. P values were calculated using Student t test.