EPO modulates hepcidin expression in a dose-dependent manner in HepG2 cells and in freshly isolated mouse hepatocytes. (A) qRT-PCR analysis of HAMP mRNA expression in HepG2 cells treated with rEPO or IL-6. Figure represents the average plus SD of 3 independent experiments; * denotes a statistically significant difference between treated samples and mock (P < .05; one-way ANOVA). (B) qRT-PCR analysis of HAMP mRNA expression in HepG2 cells treated with rEPO (1 or 2 U/mL) and/or with 0.1 to 10 μg/mL anti-EPOR. Bars on the right of each group correspond to HAMP mRNA levels after preabsorption of the anti-EPOR antibody with a specific peptide (+Block). * denotes a significant difference (P < .05) between EPO(+)/anti-EPOR(+)–treated samples and the EPO(+)/anti-EPOR(−) controls (one-way ANOVA) ** stands for a significant difference P < .05 level, between EPO(−)/anti-EPOR(+) or EPO(−)/anti-EPOR(+)/Block(+) and the EPO(−)/anti-EPOR(−) control (2-way ANOVA). Figure represents the average + SD of 3 independent experiments. (C) qRT-PCR analysis of Hamp1 mRNA expression in freshly isolated mouse hepatocytes treated with 0.01 to 2.0 U/mL rEPO (gray bars), and/or 0.1 to 5 μg anti-EPOR (white and black bars, respectively). Untreated HepG2 cells were used as control (hatched bar, left). Treatment with IL-6 was used to assess Hamp1 response to stimulation (gray bar, right). A total of 7 animals were used for each treatment; * denotes a significant difference (P < .05) between EPO(+)/anti-EPOR(−) or EPO(−)/anti-EPOR(+) or IL-6(+) and the nontreated control (one-way ANOVA). ** stands for a significant difference at the 95% confidence level between (EPO(+)/anti-EPOR(+) and the EPO(1 U)/anti-EPOR(−) control (one-way ANOVA).