Figure 2.
Arginine is required for erythroid lineage commitment and its absence attenuates terminal differentiation. (A) The fate of progenitors treated with rEPO in the presence (+) or absence (−) or exogenous arginine was monitored as a function of CD34 and CD36 expression in IL3R−GlyA− cells at day 3 of differentiation. Representative dot plots are presented (top) and IL3R−GlyA−CD34−CD36+ (CFU-E) cells were quantified relative to differentiation in the presence of arginine (bottom, n = 13). (B) Relative levels of erythroid and myeloid differentiation were monitored by GlyA and CD11b staining, respectively. Representative dot plots are shown at days 3 and 7 of differentiation in the presence or absence of arginine (top). Quantification of GlyA+ (n = 17, day 3; n = 9, day 7) and CD11b+ cells (n = 16, day 3; n = 7, day 7) are presented (bottom). (C) The impact of arginine at later stages of erythroid differentiation was evaluated by depleting arginine after 3 days of EPO-induced erythroid differentiation. GlyA was evaluated at day 3 (top histogram; gray histograms, isotype control; solid line histograms, specific staining) and then 4 days later (day 7) in the presence or absence of arginine (bottom histograms, solid line, and orange histograms, respectively). Quantification of the percentages of GlyA+ cells was compared after EPO-induced differentiation from day 3 to 7 in the presence or absence of arginine (right, n = 9). (D) The impact of arginine deprivation between days 3 and 10 of rEPO-induced differentiation was monitored as a function of CD49d/GLUT1 profiles (top) and enucleation (bottom, Syto16 staining). Representative plots are shown (left) and quantification in 8 individual donors is presented (right). (E) Arginase 2 expression was evaluated by immunoblot at days 4 and 7 of differentiation in the absence or presence of EPO. Actin levels are shown as a loading control (left). Quantification of arginase 2 expression relative to actin was evaluated in the different conditions (right, n = 3). ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .0001.

Arginine is required for erythroid lineage commitment and its absence attenuates terminal differentiation. (A) The fate of progenitors treated with rEPO in the presence (+) or absence (−) or exogenous arginine was monitored as a function of CD34 and CD36 expression in IL3R−GlyA− cells at day 3 of differentiation. Representative dot plots are presented (top) and IL3R−GlyA−CD34−CD36+ (CFU-E) cells were quantified relative to differentiation in the presence of arginine (bottom, n = 13). (B) Relative levels of erythroid and myeloid differentiation were monitored by GlyA and CD11b staining, respectively. Representative dot plots are shown at days 3 and 7 of differentiation in the presence or absence of arginine (top). Quantification of GlyA+ (n = 17, day 3; n = 9, day 7) and CD11b+ cells (n = 16, day 3; n = 7, day 7) are presented (bottom). (C) The impact of arginine at later stages of erythroid differentiation was evaluated by depleting arginine after 3 days of EPO-induced erythroid differentiation. GlyA was evaluated at day 3 (top histogram; gray histograms, isotype control; solid line histograms, specific staining) and then 4 days later (day 7) in the presence or absence of arginine (bottom histograms, solid line, and orange histograms, respectively). Quantification of the percentages of GlyA+ cells was compared after EPO-induced differentiation from day 3 to 7 in the presence or absence of arginine (right, n = 9). (D) The impact of arginine deprivation between days 3 and 10 of rEPO-induced differentiation was monitored as a function of CD49d/GLUT1 profiles (top) and enucleation (bottom, Syto16 staining). Representative plots are shown (left) and quantification in 8 individual donors is presented (right). (E) Arginase 2 expression was evaluated by immunoblot at days 4 and 7 of differentiation in the absence or presence of EPO. Actin levels are shown as a loading control (left). Quantification of arginase 2 expression relative to actin was evaluated in the different conditions (right, n = 3). ∗P < .05; ∗∗P < .01; ∗∗∗∗P < .0001.

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