Functional and cellular characterization of IFN-α2 and IFN-α16 in hematopoietic stem cells. (A-B) CD34⁺ HSCs were expanded in TPO-conditioned media supplemented (or not) with IFNs α2b (500 U/mL) or equimolar concentrations (50 ng/mL) of IFN-α16^WT or IFN-α16^MUT at 2-day intervals (A), followed by flow cytometric quantification of CD41⁺ (integrin α_IIb) and Annexin V (phosphatidylserine) at designated time points (B). Data represent the mean ± standard error of the mean (SEM) from triplicate determinations of a single representative experiment, repeated once. (C) Differentiating CD34⁺ HSCs at specific time points (day 0 [D0], D4, and D14) were stimulated with IFN-α2a (500 U/mL) or equimolar (50 ng/mL) concentrations of IFN-α16^WT or IFN-α16^Mut for 60 minutes, followed by RNA isolation and quantification of selected IRGs (IFIT1, IFITM3, IFRD1, OAS1, MX1, and ISG15) or MK-specific genes (VWF and PF4). Data are from a single representative experiment (mean ± SEM from triplicate determinations normalized to actin), repeated once; statistically significant differences (α2b to either α16^WT [red asterisks] or α16^MUT [black asterisks]) only seen at D4 are shown (t-test; **P < .01, ***P < .001). (D) D4 HSCs were stimulated for 60 minutes with IFN-α2a (500 U/mL) or equimolar (50 ng/mL) concentrations of IFN-α16^WT or IFN-α16^Mut, followed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and pSTAT1 detection by immunoblot analysis (20 μg/lane); α-tubulin and total cellular STAT1 are shown as loading controls. Heat-inactivated IFN-α16^WT and IFN-α16^MUT exhibit no pSTAT1 activation. N.S., not significant.