<p><b>(A)</b> Western blot analysis of LC3B protein of WT and <i>Foxo3</i><sup><i>-/-</i></sup> bone marrow TER119<sup>+</sup> cells (n = 3 mice for each genotype). Quantification of the LC3BII/ LC3B-I ratio in one representative of two independent experiments is shown (bottom panel). <b>(B)</b> Western blot analysis of LC3B protein extracted from WT and <i>Foxo3</i><sup><i>-/-</i></sup> bone marrow erythroblasts Gates II to IV (insufficient Gate I cell numbers for Western blot). Quantification of the LC3B-II/LC3B-I ratio is shown (panel below). <b>(C)</b> Autophagic flux in WT and <i>Foxo3</i><sup><i>-/-</i></sup> bone marrow cells was analyzed by flow cytometry. Cells were cultured with chloroquine (50 μM) for the indicated time points and autophagosomes were detected by Cyto-ID in specific gates according to TER119, CD44 and FSC properties. Flux was calculated by subtracting the value obtained from the untreated sample to the value obtained at each of the different time points. Results are mean ± SEM of n = 3. One representative of three independent experiments is shown. <b>(D)</b> Aliquots of cell lysates from (<b>C</b>) at the indicated time points were subjected to Western blot analysis of LC3B showing two replicates. Quantification of the LC3B-II protein is normalized to total actin, and the relative accumulation of LC3B-II is quantified (bottom panel). <b>(E)</b> Flow cytometry analysis (left panels) and quantification (right panel, n = 4 in each genotype) of Mitotracker Red CMXRos in combination with CD71 surface expression of WT and <i>Foxo3</i><sup><i>-/-</i></sup> peripheral blood. *<i>P</i> < 0.05 **<i>P</i> < 0.01 ***<i>P <</i> 0.001, Student’s <i>t</i> test.</p
