Figure 2.
ATG4A promotes human erythroid maturation and enucleation. (A) Human CB-derived CD34+ HSPCs were transduced with control (shLUC) or ATG4A KD (shATG4A) lentiviruses, expanded, and sorted before erythroid culture. (B) Colony-forming potential of CD34+ shRNA expressing HSPCs plated into methylcellulose-containing cytokines. Myeloid, erythroid, or multilineage (CFU-GEMM) colonies were scored by 2 independent investigators. Data shown are plotted as mean ± SEM of 3 independent donors and 2 shRNA hairpins per group. Significance was calculated using a 2-way ANOVA followed by a multiple comparisons test. (C) Fold expansion of erythroid cells stained with CD71 and CD235a on days 5 and 9 of erythroid culture. Data shown are plotted as mean ± SEM of 3 independent donors and 2-shRNA hairpins per group. (D) Erythroid cell morphology evaluated using May-Grunwald-Giemsa–stained cytospins of control or ATG4A KD cells on days 14, 16, and 18 of erythroid culture. Images were taken at 60× magnification. The scale bar is 50 µm. The arrow in the top row highlights enucleated cells in control erythroid cultures. (E) Quantification of erythroid cell morphology on days 14 and 18 May-Grunwald-Giemsa–stained cytospins from control (shLUC) cultures. Baso, basophilic erythroblast; poly, polychromatic erythroblast; ortho, orthochromatic erythroblast; retic, reticulocyte. (F) Quantification of erythroid cell morphology on days 14 and 18 May-Grunwald-Giemsa–stained cytospins from ATG4A KD (shATG4A) cultures. (G) Immunophenotypic evaluation of control and ATG4A KD cells from day 18 of erythroid culture, stained with CD49d. (H) Quantification of CD49d-positive cells on days 9, 14, 16, and 18 of erythroid culture. Data shown are plotted as mean ± SEM of 2 independent donors. Significance calculated using Student 2-tailed t test. (I and J) Enucleation efficiency on day 18 of differentiation. Enucleation efficiency was quantified using flow cytometry. Enucleated cells were defined as GLYA+ SYTO60−. Data shown are plotted as mean ± SEM of 3 independent donors and 2 shRNA hairpins per group.

ATG4A promotes human erythroid maturation and enucleation. (A) Human CB-derived CD34+ HSPCs were transduced with control (shLUC) or ATG4A KD (shATG4A) lentiviruses, expanded, and sorted before erythroid culture. (B) Colony-forming potential of CD34+ shRNA expressing HSPCs plated into methylcellulose-containing cytokines. Myeloid, erythroid, or multilineage (CFU-GEMM) colonies were scored by 2 independent investigators. Data shown are plotted as mean ± SEM of 3 independent donors and 2 shRNA hairpins per group. Significance was calculated using a 2-way ANOVA followed by a multiple comparisons test. (C) Fold expansion of erythroid cells stained with CD71 and CD235a on days 5 and 9 of erythroid culture. Data shown are plotted as mean ± SEM of 3 independent donors and 2-shRNA hairpins per group. (D) Erythroid cell morphology evaluated using May-Grunwald-Giemsa–stained cytospins of control or ATG4A KD cells on days 14, 16, and 18 of erythroid culture. Images were taken at 60× magnification. The scale bar is 50 µm. The arrow in the top row highlights enucleated cells in control erythroid cultures. (E) Quantification of erythroid cell morphology on days 14 and 18 May-Grunwald-Giemsa–stained cytospins from control (shLUC) cultures. Baso, basophilic erythroblast; poly, polychromatic erythroblast; ortho, orthochromatic erythroblast; retic, reticulocyte. (F) Quantification of erythroid cell morphology on days 14 and 18 May-Grunwald-Giemsa–stained cytospins from ATG4A KD (shATG4A) cultures. (G) Immunophenotypic evaluation of control and ATG4A KD cells from day 18 of erythroid culture, stained with CD49d. (H) Quantification of CD49d-positive cells on days 9, 14, 16, and 18 of erythroid culture. Data shown are plotted as mean ± SEM of 2 independent donors. Significance calculated using Student 2-tailed t test. (I and J) Enucleation efficiency on day 18 of differentiation. Enucleation efficiency was quantified using flow cytometry. Enucleated cells were defined as GLYA+ SYTO60−. Data shown are plotted as mean ± SEM of 3 independent donors and 2 shRNA hairpins per group.

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