DFP results in increased serum iron (A) and transferrin saturation (B) while reducing parenchymal iron in the liver, spleen, and bone marrow (C-E). While liver Hamp mRNA expression is unchanged in …
Source data for iron-related parameters in wild type (WT), myelodysplastic syndrome (MDS), and DFP-treated MDS mice.
Bone marrow erythroblasts were isolated using flow gating strategy and apoptosis was measured using activated caspase 3/7. Erythroblast apoptosis is elevated in ProE, BasoE, PolyE, and OrthoE from …
Source data for flow analysis of apoptosis measured by activated caspase 3/7 in bone marrow erythroblasts from wild type (WT) and myelodysplastic syndrome (MDS) mice.
Serum DFP concentration is measurable in DFP-treated MDS mice. (n=3–5 mice/group). WT = wild type; MDS = myelodysplastic syndrome; DFP = deferiprone.
Source data for serum deferiprone (DFP) concentration in DFP-treated wild type (WT) and myelodysplastic syndrome (MDS) mice.
Similarly elevated transferrin is observed in male and female DFP-treated MDS mice (n=3–4 male mice/group and n=4–6 female mice/group). *p<0.05 vs. control; Con = control; DFP = deferiprone; sat = …
Source data for transferrin saturation in male and female myelodysplastic syndrome (MDS) and DFP-treated MDS mice.
(A) Western blot of bone marrow CD45 negative cell protein extracts demonstrate no difference in FTH1 between WT, MDS, and DFP-treated MDS mice; the gel is quantified in (B) (n=2 mice/group, …
Western blots with ferritin H antibody staining relative to actin in bone marrow erythroblast enriched CD45 negative cells from wild type (WT), myelodysplastic syndrome (MDS), and DFP-treated MDS mice.
Source data for quantification of ferritin heavy chain (FTH1) protein concentration relative to actin in bone marrow erythroblast enriched CD45 negative cells from wild type (WT), myelodysplastic syndrome (MDS), and DFP-treated MDS mice.
Western blot of liver protein extracts demonstrate no obvious differences in STAT3 signaling (A), and no differences in Saa1 mRNA expression (B), demonstrating no change in the inflammatory …
Western blots with STAT3 and pSTAT3 antibody staining relative to GAPDH in liver from wild type (WT), myelodysplastic syndrome (MDS), and DFP-treated MDS mice.
Source data for Saa1 in liver from wild type (WT), myelodysplastic syndrome (MDS), and DFP-treated MDS mice.
Sorted bone marrow erythroblast Erfe mRNA expression between WT and DFP-treated WT mice (n=5–12 mice/group). *p<0.05 vs. WT DFP; WT = wild type; DFP = deferiprone; Erfe = erythroferrone.
Source data for erythroferrone (Erfe) in sorted bone marrow erythroblasts from wild type (WT) and DFP-treated WT mice.
Circulating RBC count (A), hemoglobin (B), MCV (C), reticulocyte count (D), platelet count (E), WBC count (F), neutrophil count (G), lymphocyte count (H), and monocyte count (i) in WT, MDS, and …
Source data for circulating cell counts and other parameters in wild type (WT), myelodysplastic syndrome (MDS), and DFP-treated MDS mice.
Similarly elevated hemoglobin is observed in male and female DFP-treated MDS mice (n=4–6 male mice/group and n=4–9 female mice/group). *p<0.05 vs. control; Con = control; DFP = deferiprone.
Source data for hemoglobin in male and female myelodysplastic syndrome (MDS) and DFP-treated MDS mice.
Spleen weight (n=11–12 mice/group) (A), splenic architecture (n=5 mice/group) (B), serum EPO concentration (n=5–12 mice/group) (C), bone marrow erythroblast count (n=13–15 mice/group) (D), and the …
Source data for erythropoiesis-related parameters in serum, bone marrow, and spleen from wild type (WT), myelodysplastic syndrome (MDS), and DFP-treated MDS mice.
Bone marrow erythroblasts are decreased to a similar degree in male and female DFP-treated MDS mice (n=4–7 male mice/group and n=8–9 female mice/group). *p<0.05 vs. control; Con = control; DFP = …
Source data for bone marrow erythroblasts in male and female myelodysplastic syndrome (MDS) and DFP-treated MDS mice.
No differences are observed in RBC count (A), hemoglobin (B), MCV (C), reticulocytes (D), or serum EPO concentration (E) from DFP-treated relative to untreated WT mice (n=5–14 mice/group). WT = wild …
Source data for circulating red blood cell parameters and serum erythropoietin from wild type (WT) and DFP-treated WT mice.
Bone marrow erythroblast fraction (n=5–13 mice/group) significantly decreased (A) particularly as a consequence of fewer BasoE, PolyE, and OrthoE (B) in DFP-treated WT mice (n=5–11 mice/group). *p<0.…
Source data for total bone marrow erythroblasts from wild type (WT) and DFP-treated WT mice.
Source data for bone marrow ProE, BasoE, PolyE, and OrthoE erythroblasts from wild type (WT) and DFP-treated WT mice.
Erythroblast apoptosis, as measured by activated caspase 3 and 7 (n=6–9 mice/group) (A), and ROS (B) are unchanged in DFP-treated relative to untreated WT mice (n=5–9 mice/group). WT = wild type; …
Source data for bone marrow erythroblast apoptosis as measured by activated caspase 3/7 and ROS from wild type (WT) and DFP-treated WT mice.
Serum concentration of DFP and of its metabolite, DFP-G, is significantly lower in DFP-treated MDS relative to DFP-treated WT mice (n=5 mice/group). *p<0.05 vs. WT DFP; &&p<0.01 vs. WT DFP-G; WT = …
Source data for serum DFP and G-DFP concentrations from DFP-treated wild type (WT) and MDS mice.
Mouse bone marrow is flushed from the femur, processed to remove debris, and filtered to collect CD45 negative flow-through cells. (A) After staining with all required antibodies, CD45-/CD11b-/Gr1- …
Gata1 mRNA expression is increased in sorted bone marrow ProE (A), unchanged in BasoE (B), and significantly decreased in PolyE (C) erythroblasts from MDS relative to WT mice; DFP treatment restores …
Source data for gene expression downstream of erythropoietin (EPO) in sorted bone marrow erythroblasts from wild type (WT), myelodysplastic syndrome (MDS), and DFP-treated MDS mice.
Sorted bone marrow erythroblast Bcl11a mRNA expression between WT and DFP-treated WT mice (n=5–12 mice/group). *p<0.05 vs. WT DFP; WT = wild type; DFP = deferiprone; Bcl11a = B cell lymphoma 11 a, …
Source data for B cell lymphoma (Bcl11a) in sorted bone marrow erythroblasts from wild type (WT) and DFP-treated WT mice.
(A) Western blot of bone marrow CD45 negative cell protein extracts demonstrate no differences in STAT5 signaling between untreated and DFP-treated MDS mice, quantified in (B). Similarly, AKT …
Western blots with pSTAT5, STAT5, pAKT, and AKT antibody staining relative to actin in bone marrow erythroblast-enriched CD45 negative cells from myelodysplastic syndrome (MDS) and DFP-treated MDS mice.
Source data for quantification of signaling via STAT5 and AKT in bone marrow erythroblast-enriched CD45 negative cells from myelodysplastic syndrome (MDS) and DFP-treated MDS mice.
DFP treatment in MDS mice increases Epor mRNA expression in sorted bone marrow following ProE (A), namely in BasoE (B), PolyE (C), and OrthoE (D) erythroblasts relative to untreated MDS or WT mice. …
Source data for erythropoietin receptor (Epor) in sorted bone marrow erythroblasts from wild type (WT), myelodysplastic syndrome (MDS), and DFP-treated MDS mice.
Sorted bone marrow erythroblast Epor mRNA expression between WT and DFP-treated WT mice (n=5–10 mice/group). *<0.05 vs. WT DFP; WT = wild type; DFP = deferiprone; Epor = erythropoietin receptor.
Source data for erythropoietin receptor (Epor) in sorted bone marrow erythroblasts from wild type (WT) and DFP-treated WT mice.
(A) DFP restores erythroblast enucleation (n=5–7 mice/group) in MDS mouse bone marrow, quantified in (B) as the fraction of enucleated relative to the total of nucleated and enucleated …
Source data for flow analysis of enucleation in erythroblasts from wild type (WT), myelodysplastic syndrome (MDS), and DFP-treated MDS mice.
(A) Flow cytometry gating using TER119 and CD44 was used to delineate bone marrow erythroblasts. In these gated erythroblasts, we evaluate membrane TFR1 to demonstrate its decrease in response to …
Source data for flow analysis of cell surface TFR1 and Tfrc in sorted bone marrow erythroblasts from wild type (WT), myelodysplastic syndrome (MDS), and DFP-treated MDS mice.
Membrane TFR1 on bone marrow erythroblasts from WT and DFP-treated WT mice (n=5–11 mice/group). *p<0.05 vs. WT DFP; WT = wild type; DFP = deferiprone; TFR1 = transferrin receptor 1; MFI = mean …
Source data for flow analysis of cell surface transferrin receptor 1 (TFR1) on bone marrow erythroblasts from wild type (WT) and DFP-treated WT mice.
Sorted bone marrow erythroblast Tfrc mRNA expression between WT and DFP-treated WT mice (n=5–7 mice/group). *p<0.05 vs. WT DFP; WT = wild type; DFP = deferiprone; Tfrc = transferrin receptor 1.
Source data for Tfrc in sorted bone marrow erythroblasts from wild type (WT) and DFP-treated WT mice.
(A) Flow cytometry gating using TER119 and CD44 was used to delineate bone marrow erythroblasts. In these gated erythroblasts, we evaluate membrane TFR2 which is unchanged in MDS and DFP-treated MDS …
Source data for flow analysis of cell surface transferrin receptor 2 (TFR2), TFR2 protein concentration in bone marrow erythroblast-enriched CD45 negative cells, and TFR2, in sorted bone marrow erythroblasts from wild type (WT), myelodysplastic syndrome(MDS), and DFP-treated MDS mice.
Western blots with transferrin receptor 2 (TFR2) antibody staining relative to actin in bone marrow erythroblast enriched CD45 negative cells from wild type (WT), myelodysplastic syndrome (MDS), and DFP-treated MDS mice.
No differences are observed in TFR2 mRNA expression in sorted BasoE (A), PolyE (B), and OrthoE (C) relative to ProE in WT, MDS, and DFP-treated MDS mice (n=5–15 mice/group). WT = wild type; MDS = …
Source data for transferrin receptor 2 (TFR2) in sorted bone marrow BasoE, PolyE, and OrthoE from wild type (WT), myelodysplastic syndrome (MDS), and DFP-treated MDS mice.
No differences in Scrib mRNA expression are observed in sorted bone marrow ProE (A), BasoE (B), PolyE (C), and OrthoE (D) between WT, MDS, and DFP-treated MDS mice (n=15–18 mice/group). WT = wild …
Source data for Scribble (Scrib) in sorted bone marrow erythroblasts from wild type (WT), myelodysplastic syndrome (MDS), and DFP-treated MDS mice.
Sorted bone marrow Pcbp1 mRNA expression is increased in ProE (A) from MDS relative to WT mice and remains high or is further elevated in ProE, BasoE (B), and PolyE (C) DFP-treated relative to …
Source data for Pcbp1 and Ncoa4 in sorted bone marrow erythroblasts from wild type (WT), myelodysplastic syndrome (MDS), and DFP-treated MDS mice.
Pcbp2 mRNA expression changes parallel those of Pcbp1 in sorted bone marrow ProE (A), BasoE (B), PolyE (C), and OrthoE (d) between WT, MDS, and DFP-treated MDS mice (n=15–18 mice / group). *p<0.05 …
Source data for Pcbp2 in sorted bone marrow erythroblasts from wild type (WT), myelodysplastic syndrome (MDS), and DFP-treated MDS mice.
No differences in Gpx4 mRNA expression are observed in sorted bone marrow ProE (A), BasoE (B), PolyE (C), and OrthoE (D) between WT, MDS, and DFP-treated MDS mice (n=15–18 mice/group). WT = wild …
Source data for glutathione peroxidase 4 (Gpx4) in sorted bone marrow erythroblasts from wild type (WT), myelodysplastic syndrome (MDS), and DFP-treated MDS mice.
Increased expression of Tfrc (A), Epor (B), Gata1 (C), Bcl2l1 (D), and Erfe (E) is expected and validates the database. No difference in Pcbp1 (F), borderline increase in Pcbp2 (G), and …
Source data for iron metabolism-related genes in bone marrow stem and progenitor cells from myelodysplastic syndrome (MDS) patients vs. controls.
Erythroblast iron uptake is mediated by TFR1 via endocytosis of clathrin-coated pits. In addition, erythroblast ferritin iron delivery is an obligatory step in erythropoiesis, and chaperones, i.e., …
RBC count | Hb | MCV | Retic count | WBC count | Platelet count | Serum EPO | Bone marrow cells | Liver iron concentration | |
---|---|---|---|---|---|---|---|---|---|
(units) | (106 /μL) | (g/dL) | (fL) | (106 /μL) | (106 /μL) | (103 /μL) | (μg/μL) | (107 cells) | (mg/g dry weight) |
WT | 10±0.1 | 14.7±0.1 | 50±0.5 | 495±67 | 5.5±0.6 | 759±67 | 308±48 | 12.4±0.7 | 0.23±0.07 |
MDS | 6.73±0.29 | 11.2±0.4 | 60±1.3 | 440±30 | 3.0±0.4 | 672±80 | 4832±1,154 | 15±0.8 | 0.51±0.04 |
*** | *** | *** | NS | ** | NS | *** | * | * |
WT = wild type; MDS = myelodysplastic syndrome (NHD13) mice; RBC = red blood cell; Hb = hemoglobin; MCV = mean corpuscular hemoglobin; Retic = reticulocyte; WBC = white blood cell; EPO = erythropoietin; NS = not significant; * P<0.05; ** P<0.01; *** P<0.0001.
Tables of reagents used for PCR and western blot experiments.
(a) Table of primers. (b) Table of antibodies.