Fig. 2

AUR upregulates hepcidin expression by activating the STAT3 pathway. aHAMP1 mRNA was measured in Huh7 cells pretreated with Stattic (10 μM), LDN-193189 (150 nM), or U0126 (10 μM) for 1 h, followed by an additional 18 h in the presence or absence of AUR (0.5 μM). bHAMP1 mRNA was measured in Huh7 cells treated with 50 ng/ml BMP6 or 50 ng/ml IL-6 in the presence or absence of AUR (0.5 μM) for 18 h. The mRNA levels were normalized to β-ACTIN and are expressed relative to the mean control value. c P-SMAD1/5/8, SMAD1, P-STAT3, STAT3, P-ERK1/2, ERK1/2, and β-ACTIN proteins were measured in untreated Huh7 cells (DMSO) and cells treated with BMP6 (50 ng/ml) or AUR for 12 h (left panel), or in Huh7 cells treated with 0.5 μM auranofin for the indicated times (right panel). d P-SMAD1/5/8, SMAD1, P-STAT3, STAT3, P-ERK1/2, ERK1/2, and β-ACTIN were measured in untreated Huh7 cells and cells treated with 50 ng/ml BMP6, 0.5 μM AUR, 10 μM Stattic, or 150 nM LDN-193189; where indicated, the cells were pretreated with Stattic or LDN, followed by AUR for an additional 18 h. For the protein quantification, P-STAT3/STAT3 and P-ERK1/2/ERK1/2 were normalized to their respective untreated groups. The cell line based experiments were repeated three independent times. Error bars indicate the SEM. The data in a were analyzed using the Student’s t-test (*p < 0.05, **p < 0.01, and N.S., not significant). The data in b and d were analyzed using a one-way ANOVA with Tukey’s post hoc test; groups labeled without a common letter were significantly different (p < 0.05)