FOXOs have no impact on O2^.− levels of HSPCs. (A) Kaplan-Meier survival curves of patients with CN-AML (GSE12417) based on FOXO3 and FOXO1 gene expression levels using median expression value cutoff. Log-rank (Mantel-Cox) test was used to determine statistical significance between groups. (B-C) MV4-11 AML cells were transduced with the indicated GFP- and shRNA-encoding lentiviral vector, or were left untreated (−). After 3 days, (B) protein expression was analyzed via western blot, using the indicated antibodies; β-actin was used as loading control. (C) Alternatively, oxidative stress was assessed in viable GFP⁺ cells using a DHE probe and flow cytometry. (D-E) CB CD34⁺ HSPCs were either transduced with the indicated GFP- and shRNA-encoding lentiviral vector and maintained in culture for 3 days (D), or maintained in culture for 3 days and incubated with the indicated drug during the last 18 hours (E). O₂^.−levels of CD34⁺ cells were then assessed using a DHE probe and flow cytometry in transduced GFP⁺ (D) or total (D,E) cells. H₂O₂ (350 μM) was used as a positive control; data are expressed as MFI (a.u.). H₂O₂, hydrogen peroxide.