Figure 2.
Abnormal erythropoiesis in Rpl11+/▵ mice and L-leucine treatment. (A) Burst-forming unit-erythroid (BFU-E) colony counts. BM cells (2 ×105) in 1 mL of MethoCult M3234 medium supplemented with 1 or 10 units/mL recombinant mouse EPO. Number of BFU-E colonies (containing >10 cells) was counted on days 8 to 10 using STEMvision (STEMCELL Technologies). N = 5 per group. (B) Frequency analysis of hematopoietic stem cells/progenitor cells by flow cytometry. Fresh single BM cells were collected from the femurs and tibias. LIN−, lineage negative (CD4–CD8–B220-Gr1-CD11b–Ter119–); LSK, LIN– Scal-1+ c-Kit+; hematopoietic stem cells, LIN-Scal1+cKit+CD48–CD150+, multipotent progenitors, LIN-Scal1+cKit+CD48–CD150–. N = 5 per group. (C) Representative flow cytometry analysis of erythroid differentiation trajectory in BM cells from Rpl11+/▵ mice and WT littermates. LIN− population (CD4–CD8–B220-Gr1-CD11b–) was analyzed and gated as previously reported16 to identify the LNPCs and erythroblast populations I to V, which include BFU-E deriving cells and proerythroblasts (I), basophilic erythroblasts (II), polychromatic erythroblasts (III), orthochromatic erythroblasts (IV), and reticulocytes and mature RBCs (V). The data revealed a differentiation block of BFU-E-derived cells/proerythroblasts (I-II) before polychromatic erythroblasts (III). (D) Frequency analysis of flow cytometry data for the populations of erythroid differentiation trajectory in the BM from Rpl11+/▵ mice and WT littermates. N = 7 per group. (E) Mice treated with 1.5% l-leucine in drinking water at the age of 4 to 6 weeks for 16 weeks. Blood Hgb concentration was monitored every 4 weeks. (F) Kaplan-Meier analysis shows that L-leucine treatment significantly prolonged the survival of the treated group (P < .05). Statistical differences between groups were calculated using a 2-tailed Student t test. Data are presented as mean ± standard error of the mean, ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. More supportive data are provided in supplemental Figures 2 and 3. FSC, forward scatter; LNPC, LIN− precursor cells; wks, weeks.

Abnormal erythropoiesis in Rpl11+/▵ mice and L-leucine treatment. (A) Burst-forming unit-erythroid (BFU-E) colony counts. BM cells (2 ×105) in 1 mL of MethoCult M3234 medium supplemented with 1 or 10 units/mL recombinant mouse EPO. Number of BFU-E colonies (containing >10 cells) was counted on days 8 to 10 using STEMvision (STEMCELL Technologies). N = 5 per group. (B) Frequency analysis of hematopoietic stem cells/progenitor cells by flow cytometry. Fresh single BM cells were collected from the femurs and tibias. LIN−, lineage negative (CD4–CD8–B220-Gr1-CD11b–Ter119–); LSK, LIN– Scal-1+ c-Kit+; hematopoietic stem cells, LIN-Scal1+cKit+CD48–CD150+, multipotent progenitors, LIN-Scal1+cKit+CD48–CD150–. N = 5 per group. (C) Representative flow cytometry analysis of erythroid differentiation trajectory in BM cells from Rpl11+/▵ mice and WT littermates. LIN− population (CD4–CD8–B220-Gr1-CD11b–) was analyzed and gated as previously reported16 to identify the LNPCs and erythroblast populations I to V, which include BFU-E deriving cells and proerythroblasts (I), basophilic erythroblasts (II), polychromatic erythroblasts (III), orthochromatic erythroblasts (IV), and reticulocytes and mature RBCs (V). The data revealed a differentiation block of BFU-E-derived cells/proerythroblasts (I-II) before polychromatic erythroblasts (III). (D) Frequency analysis of flow cytometry data for the populations of erythroid differentiation trajectory in the BM from Rpl11+/▵ mice and WT littermates. N = 7 per group. (E) Mice treated with 1.5% l-leucine in drinking water at the age of 4 to 6 weeks for 16 weeks. Blood Hgb concentration was monitored every 4 weeks. (F) Kaplan-Meier analysis shows that L-leucine treatment significantly prolonged the survival of the treated group (P < .05). Statistical differences between groups were calculated using a 2-tailed Student t test. Data are presented as mean ± standard error of the mean, ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. More supportive data are provided in supplemental Figures 2 and 3. FSC, forward scatter; LNPC, LIN− precursor cells; wks, weeks.

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