Fig 2: IgJCreERT2 mice were immunized with SRBCs. On day 5, the A. spleen and B. bone marrow were collected and GFP+ live CD45+ singlets were evaluated for their expression of GFP, CD138, CD19, CD44, CD98, MHCII, CD69 and CD93 and compared to B2 cells, defined as CD19/B220+ cells.

Fig 3: SLC7A5/SLC3A2 expression in circulating CD71HI/Ter119HI reticulocytes under anemic conditions and in response to rhEPO(A) Representative flow cytometry analysis showing circulating CD71HI/Ter119HI reticulocytes in PHZ-treated and control mice, as well as in mice subjected to phlebotomy. For PHZ a representative experiment out of 20 is shown. For phlebotomy, a representative experiment out of 8 is shown.(B) Representative histograms of SLC3A2 expression in circulating CD71HI/Ter119HI reticulocytes of untreated control mice (in blue), mice treated with PHZ or mice subjected to phlebotomy (in red), as well as ErGFPcre Slc7a5LoxP/LoxP mice treated with PHZ or subjected to phlebotomy (in green). The IgG-APC control is represented in gray. Quantification of the mean fluorescence intensity (MFI) of SLC3A2 on circulating CD71HI/Ter119HI reticulocytes from control untreated mice (n = 33), control mice treated with PHZ (n = 20), ErGFPcre Slc7a5LoxP/+ untreated mice (n = 11), ErGFPcre Slc7a5LoxP/+ mice treated with PHZ (n = 10), ErGFPcre Slc7a5LoxP/LoxP untreated mice (n = 16) and ErGFPcre Slc7a5LoxP/LoxP mice treated with PHZ (n = 14). Quantification of the mean fluorescence intensity (MFI) of SLC3A2 on circulating CD71HI/Ter119HI reticulocytes from control mice (n = 8), control mice subjected to phlebotomy (n = 8), ErGFPcre Slc7a5LoxP/+ untreated mice (n = 4), ErGFPcre Slc7a5LoxP/+ mice subjected to phlebotomy (n = 4), ErGFPcre Slc7a5LoxP/LoxP untreated mice (n = 3) and ErGFPcre Slc7a5LoxP/LoxP mice subjected to phlebotomy (n = 3).(C) Western blot analyses of SLC7A5 relative to β-actin protein levels in circulating erythroid cells of control and ErGFPcre Slc7a5LoxP/LoxP PHZ-treated mice.(D) L-phenylalanine uptake in isolated CD71HI/Ter119HI circulating reticulocytes from PHZ-treated control or ErGFPcre Slc7a5LoxP/LoxP mice (n = 5).(E) Representative flow cytometry analysis of CD71 expression versus GYPA in healthy and anemic subjects (left panels). Histograms of SLC3A2 expression in circulating CD71HI/GYPAHI and CD71NEG/GYPAHI populations of control (blue) and anemic (red) subjects (right panels). A representative experiment out of 3 anemic subjects and 3 control subjects is shown.(F) Representative histograms of SLC3A2 expression in circulating CD71HI/Ter119HI reticulocytes of untreated control mice (blue) or mice treated with rhEPO (200U) (red). The IgG-APC control is represented in gray. Quantification of the mean fluorescence intensity (MFI) of SLC3A2 on circulating CD71HI/Ter119HI reticulocytes from untreated (n = 5) or rhEPO-treated control mice (n = 5), as well as from untreated (n = 3) or rhEPO-treated ErGFPcre Slc7a5LoxP/LoxP mice (n = 5).Data are shown as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey’s post hoc test when comparing more than two groups or two-tailed Student’s t test with Welch’s correction when comparing two groups (∗∗p < 0.01; ∗∗∗p < 0.005).

Fig 4: Basal expression of SLC7A5/SLC3A2 in the erythroid lineage(A) The panel represents the flow cytometry analysis showing the different splenic cell populations in basal conditions based on the surface expression of CD71 and Ter119 in control mice. SLC3A2 expression is also represented by a color code: yellow-orange representing the strongest expression and dark green-blue the weakest or absence of expression.(B) Histograms showing SLC3A2 expression in the different splenic CD71HI/Ter119NEG/LOW, CD71HI/Ter119HI, CD71NEG/Ter119HI and CD71NEG/LOW/Ter119NEG cell populations in control (in blue) and ErGFPcre Slc7a5LoxP/LoxP mice (in green). The IgG control (in gray) is included. A representative experiment out of 23 is shown.