Fig 2: Reduced sulfation of HS in Ndst1f/fAlbCre+ decreases Hamp mRNA and plasma hepcidin after LPS injection. Ndst1f/fAlbCre+ and Ndst1f/fAlbCre- mice were fed an iron-balanced diet for 1 week and then treated with a single intraperitoneal dose of LPS (1 mg/kg). After 6 h, mice were sacrificed. A, liver Socs3 mRNA level was measured and normalized to Tbp mRNA. B, Hamp mRNA was measured in the liver and normalized to Tbp mRNA. C, serum hepcidin was measured before and after LPS treatment. D–F, serum iron (D), nonheme liver iron (E), and nonheme spleen iron (F) were measured by a spectrophotometric assay after PBS or LPS injection. G, hepatic pSTAT3 and pSMAD5 were measured by Western blotting. After quantitation of the bands by Image J, the values were normalized to STAT3 and SMAD5, respectively. H, Hep3B WT and NDST1-/- cells were transfected with pGL2Hamp-luciferase and pGL2TK-Renilla plasmid along with scrambled siRNA and STAT3 siRNA and stimulated with 50 ng/ml of IL6 for 6 h. Luciferase and Renilla activity were measured. I, Hep3B WT and NDST1-/- cells were transfected with pGL2Hamp-luciferase and pGL2TK-Renilla plasmid along with scrambled siRNA and SMAD5 siRNA and stimulated with 50 ng/ml of BMP6 for 6 h. Luciferase and Renilla activity were measured. n = 2 biological experiment done in triplicate. The data in each of the panels were analyzed by two-way ANOVA with post hoc Bonferroni multiple comparison test.

Fig 3: Reduction of HAMP expression and pSMAD5 in primary hepatocytes derived from Ndst1f/fAlbCre+ mice. A, level of Ndst1 mRNA in hepatocytes derived from Ndst1f/fAlbCre- (control) and Ndst1f/fAlbCre+ (mutant) mice. B, level of NDST1 protein. C, level of Hamp mRNA. D, SMAD5 phosphorylation in primary hepatocytes derived from Ndst1f/fAlbCre- and Ndst1f/fAlbCre+ mice treated with different concentrations of BMP6 for 6 h. Densitometry was performed using ImageJ and the values were normalized to SMAD5 as indicated (n = 2).