Treatment of HSPCs with metformin (Met) increases FOXO3 expression, increases the active form of FOXO3, and causes FOXO3 to accumulate in the nucleus. (A) Effect of Met treatment on FOXO3 expression, as measured by RT-qPCR. HSPCs derived from patients with SCD were treated on day 7 of culture and analyzed on day 14. (B) Met treatment increases the active form of FOXO3 activator AMPK (Thr172 phosphorylated AMPK) and the active nuclear-localizing form of FOXO3 (Ser413 p-FOXO3) and decreases the active form of the FOXO3 inhibitor, AKT (Ser473 phosphorylated AKT), and the cytoplasmic form of FOXO3 (Ser253 p-FOXO3). HSPCs derived from patients with SCD were treated on day 13 of culture and analyzed 24 hours later on day 14. (C) Aggregate of 3 individual western blots from 3 experiments with unique CD34⁺ donors with SCD, quantified. (D) Met treatment causes FOXO3 to accumulate in the nucleus. HSPCs from normal donors were treated with various concentrations of Met for 24 hours, collected, separated into nuclear and cytoplasmic fractions, and analyzed by western blot. Error bar represents SD. ***P < .0001, **P < .005, *P < .01. N.S, not significant.