Altered FOXO response to oxidative stress in obesity-primed HSCs. (A-B) Representative images (left) of immunofluorescence analysis of FOXO3 (A) and FOXO4 (B) subcellular localization in HSC-SLAMs, isolated from 4-month-old control and db mice, after 30 minutes in vitro treatment with medium or ROS (H₂O₂: 100 µM). Scale bar, 10 µm. Graphs (right) indicate the mean percentage ± standard deviation (SD) of HSC-SLAMs presenting FOXO3 and FOXO4 nuclear localization (n = 3-5 with more than 50 individual cells analyzed in each condition). Two-way ANOVA with Sidak’s post hoc tes; ****P ≤ .0001. (C-D) Mean percentage ± SD of control and db HSC-SLAMs presenting FOXO3 (C) and FOXO4 (D) nuclear localization after 30 minutes of in vitro treatment with stem cell factor and thrombopoietin (20 ng/mL) (n = 3 with more than 50 individual cells analyzed in each condition). Two-way ANOVA with Sidak’s post hoc test; **P ≤ .01. (E) Representative FACS histogram (left panel) and relative average mean fluorescence intensity ± SD (right panel) showing levels of intracellular ROS detected by 2′,7′-dichlorofluorescin diacetate staining in HSC-SLAMs isolated from 4-month-old control and db mice after 30 minutes of in vitro treatment with medium or ROS (H₂O_2: 100 µM) (n = 6). Two-way ANOVA with Tukey’s post hoc test; *P ≤ .05; ***P ≤ .0005. (F) Impact of ROS intensity and duration. Mean percentage ± SD of control and db HSC-SLAMs presenting FOXO3 nuclear localization after 30 minutes of in vitro treatment with medium or ROS (H₂O_2: 100 µM) (n = 3). Two-way ANOVA with Sidak’s post hoc test; ***P ≤ .001. (G-H) Mean percentage ± SD of HSC-SLAMs presenting FOXO nuclear localization when isolated from juvenile 5-week-old db mice (before obesity onset) (n = 2) (G) or 4-month-old Nkx2.1-CRE::LepR^fl/fl obese mice with neuron-specific Lepr deletion (n = 3) (H) and treated for 30 minutes in vitro with medium or ROS (H₂O₂: 100 µM). Two-way ANOVA with Sidak’s post hoc test; ****P ≤ .0001.