ATG4A promotes mitochondrial clearance during terminal erythroid maturation. (A) Scheme of the MT-Keima mitophagy reporter. (B) The 561/458 MT-Keima fluorescence ratio measured by flow cytometry on days 1, 3, 5, 9, 13, and 18 of erythroid culture. HSPCs transduced with MT-Keima control (shLUC) or MT-Keima ATG4A KD (shATG4A) reporters were stained with CD71 and GLYA to gate on a total erythroid population. A significant difference in the 561:458 ratio between control and ATG4A KD cells was found only on day 18 of culture. Data shown are plotted as mean ± SEM of 3 independent donors and analyzed using a 2-way ANOVA followed by a multiple comparisons test. (C) Representative flow plots of MT-Keima fluorescence at 458 nm or 561 nm excitation on day 18 of erythroid differentiation. (D and E) Mitotracker Deep Red (MDR) or CellROX Deep Red levels on day 18 of erythroid culture. Control and ATG4A KD erythroid cells were stained with GLYA, and MDR mean fluorescence intensity was measured by flow cytometry. Significance was evaluated using Student t test. Data shown are plotted as mean ± SEM of 3 independent donors and 2 shRNA hairpins per group (D). Data shown are plotted as mean ± SEM of 2 independent donors (E).