ATG4A promotes macroautophagy during erythropoiesis. (A) Schematic design of the LC3B-GFP-mCherry autophagy ratiometric reporter. (B) Representative histograms of mCherry and GFP fluorescence measured by flow cytometry in human erythroid cells expressing the LC3B-ratiometric reporter on days 5, 9, 12, and 18 of erythroid differentiation. (C) LC3B-ratiometric reporter mCherry/GFP ratio in control and ATG4A KD erythroid differentiation. The ratio of mCherry/GFP from control (shLUC) and ATG4A KD (shATG4A) erythroid cells was plotted on days 1, 5, 9, 12, and 18 of erythroid culture. The fluorescence of mCherry and GFP was quantified using flow cytometry. Data shown are plotted as mean ± SEM of 3 independent donors and 2 shRNA hairpins per group. Significance was determined using a 2-way ANOVA followed by a multiple comparisons test. (D) LC3B protein level in lysates harvested from days 5 and 18 of erythroid culture immunoblotted for LC3B and β-actin. (E and F) Quantification of immunoblots for LC3B-I and LC3B-II relative to β-actin on days 5 and 18 of erythroid culture. Data plotted as mean ± SEM of 2 hairpins per group from a single CB donor. (G and H) LC3B protein level in control and ATG4A KD cells from days 5 and 18 of culture treated with 200 nM of bafilomycin A1 for 4 hours. Lysates were analyzed with anti-LC3B and GAPDH. (I) The ratio of mCherry to GFP fluorescence in erythroid cells expressing the LC3B-ratiometric reporter from day 5 of culture treated with 200 nM of bafilomycin A1 for 4 hours. Following treatment, the ratio of mCherry to GFP was quantified by flow cytometry. Data shown are plotted as mean ± SEM of 3 independent donors and 2 shRNA hairpins per group. Significance was evaluated using a 2-way ANOVA followed by a multiple comparisons test.