In vitro high glucose condition alters FOXO protein function in Hoxb8-FL cells. (A-B) Experimental scheme and Hoxb8-FL cell line growth condition in low (11.1 mM) and high (55 mM) glucose media. Mean percentage ± standard deviation (SD) of the relative cell number counted (A) and cell cycle analysis (B) measured by Hoechst 33342/Ki67 staining after 3-day culture. (C) Relative average mean fluorescence intensity ± SD showing levels of intracellular ROS detected by DCFDA in Hoxb8-FL cells cultured in low and high glucose condition at steady state or upon oxidative challenge (30 minutes; H₂O₂: 100 µM). (D) Immunoblot analysis of signaling proteins in 6 × 10⁵ Hoxb8-FL cells, cultured in low and high glucose condition. Immunoblots are representative of 2 independent experiments. (E) Representative images of immunofluorescence analysis of FOXO3 subcellular localization in Hoxb8-FL cell line culture in low and high glucose condition after 30 minutes of in vitro treatment with medium or ROS (H₂O₂: 100 µM). Scale bar, 10 µm. Graph (right) indicates the mean percentage ± SD of Hoxb8-FL cells presenting FOXO3 nuclear localization (n = 3 with more than 50 individual cells analyzed in each condition). Two-way ANOVA with Sidak’s post hoc test; ****P ≤ .0001. (F-G) In vitro culture of low and high glucose adapted Hoxb8-FL cells in granulo-macrophagic differentiation condition. (F) Cell numbers over time. (G) Results are expressed as mean cell numbers ± SD. Level of apoptosis in low and high glucose adapted Hoxb8-FL cell, 6 hours after switching from self-renewing to differentiation culture conditions. Results are expressed as mean of annexin V⁺ cells ± SD (n = 4). Unpaired, 2-tailed Student t test; *P < .05; ****P < .0001. (H) In vivo differentiation of low and high glucose adapted Hoxb8-FL cells. Mean percentage of chimerism ± SD in the PB of lethally irradiated mice transplanted with Hoxb8-FL cells (n = 4). GM-CSF, granulocyte-macrophage colony-stimulating factor. Two-way ANOVA with Sidak’s post hoc test; ****P ≤ .0001.