PMC

(A) Real-time PCR analysis of Vhl, Vegf, Epo, Hamp1, Dmt1, and Trfc expression in Vhl/Hif1a/Hif2a^–/– livers. Relative mRNA expression levels were normalized to 18S ribosomal RNA. Bars represent arithmetic mean values ± SEM (n = 3). (B) Serum Epo concentrations and serum iron levels in Vhl/Hif1a/Hif2a^–/– mice (n = 3). Circles and squares represent data points for individual mice. Error bars represent SEM. *P < 0.05; **P< 0.01, for comparisons with controls.

(A) Hif-1α and Hif-2α protein levels in Phd2^–/– livers. Ponceau staining is used to assess for equal protein loading. +Co, positive control sample obtained from Vhl^–/– livers. (B) Hepatocyte-specific inactivation of Phd2 does not increase Epo mRNA and does not suppress Hamp1 mRNA levels in Phd2^–/– livers. Shown are relative mRNA expression levels normalized to 18S ribosomal RNA in mutant and control livers. Corresponding renal Epo mRNA levels are shown for comparison (n = 3). (C) Hct, reticulocyte counts, and serum Epo and serum iron levels in control and Phd2 mutant mice (n = 3 each). Shown are mean values ± SEM. For statistical analysis, mutants were compared with controls.

Gdf15 mRNA levels in total spleen and BM cell isolates and corresponding serum Gdf15 levels in pg/ml. (A) Left panels, Vhl^–/– mutants and Cre^– littermate controls (n = 3 and 4, respectively for mRNA analysis; for serum analysis, n = 4 each); middle panels, Vhl/Epo^–/– mice and Cre^– littermate controls (n = 4 each); right panels, WT mice treated with rhEPO or with vehicle (for mRNA analysis, n = 3 and 4, respectively; for serum analysis, n = 6 and 8, respectively). Shown are mean values ± SEM: *P < 0.05; **P< 0.01; ***P < 0.001, for comparisons of mutants with controls. (B) Schematic depiction of Hif’s role in the regulation of hepcidin transcription in hepatocytes. C^–, Cre-negative littermate control mice or vehicle-treated WT mice.

(A) Renal and hepatic Epo (n = 3, 5, and 3, respectively) in control mice and Vhl^–/– mutants with or without Cp treatment and liver Hamp1 RNA levels (n = 6, 4, and 3, respectively) in nontreated control, Cp-treated control, and Cp-treated Vhl^–/– mutants. Lower panels show Hct, reticulocyte counts, (n = 3 and 4, respectively), serum Epo (n = 3 and 5, respectively), and spleen to body weight ratios in nontreated control and Cp-treated Vhl^–/– mice (n = 3 each). (B) Hamp1 mRNA levels in control and Hif2a/Pax3-cre (P3) mutants exposed to chronic hypoxia (10% O₂ for 10 days) (n = 3 each) and in thalassemic mice (th3/th3) and control littermates (+/+) (n = 4 each). Shown are mean values ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001, for comparisons with control group or comparison with normoxia. Cp, mice pretreated with Cp; Hx, treatment with 10% O₂ for 10 days.

(A). Hepatic Vhl and Hamp1 mRNA levels in control, Vhl/Epo^–/–, and Vhl/Epo^–/– mice treated with rhEPO (n = 6, 6, and 5, respectively). Right panel shows Hif-1α and Hif-2α protein levels in Vhl/Epo^–/– livers. Ponceau staining is used to assess for equal protein loading. (B) Epo levels in control, Vhl/Epo^–/–, and Vhl^–/– livers and kidneys (n = 6, 6, and 3, respectively). Bottom panel shows serum Epo concentrations in control, Vhl/Epo^–/–, and Vhl/Epo^–/– mice treated with rhEPO (n = 10, 6, and 4, respectively). (C) Hct and reticulocyte counts in control, Vhl/Epo^–/–, and rhEPO-treated Vhl/Epo^–/– mice and representative FACS analysis plot of CD71/Ter119-stained BM and spleen cells from 1 control and 1 mutant mouse. Percentages of CD71^hiTer119^hi-positive cells are indicated in the right upper quadrant. (D) Liver (n = 8, 4, and 5, respectively) and serum iron concentrations (n = 10, 6, and 4, respectively) in control, Vhl/Epo^–/–, and Vhl/Epo^–/– mice treated with rhEPO, and H-ferritin protein levels in control and Vhl/Epo^–/– livers. β-actin served as loading control. Shown are mean values ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001, for comparisons of mutants with controls. Vhl/Epo^–/– (rhEPO), Vhl/Epo double-mutant mice treated with rhEPO.

(A) Shown are results from real-time PCR analysis of Vhl and Vegf mRNA levels in Vhl^–/– livers (n = 3) and Epo mRNA levels in Vhl^–/– kidneys and livers (n = 3); analysis was performed on day 8 after the first tamoxifen injection. Relative mRNA expression levels were normalized to 18S ribosomal RNA. (B) Global inactivation of Vhl induces erythropoiesis. Shown are individual Hct values (n = 14 and 13, respectively), reticulocyte counts (%) (n = 6 each), and serum Epo concentrations (sEpo) (n = 3 each) from control and mutant mice and a representative picture of a control and a Vhl^–/– spleen. Lower right panel shows a representative FACS plot of CD71/Ter119 double-stained BM and spleen cells from an individual control mouse and Vhl mutant. Percentages of CD71^hi/Ter119^hi-positive cells (right upper quadrant) are indicated. (C) Shown are relative expression levels of Hamp1 mRNA in control and Vhl^–/– livers (n = 5 and 3, respectively) and serum iron (n = 3 each) and liver iron concentrations (n = 7 and 4, respectively). H-ferritin protein levels in control and Vhl^–/– livers were determined by immunoblot in 3 mice, β-actin served as loading control. Asterisks indicate a statistically significant difference when comparisons were made to the control group: *P < 0.05; **P< 0.01; ***P< 0.001. Shown are arithmetic mean values ± SEM. Co, Cre-negative littermate control; retic, reticulocytes.