Iron induces the nuclear translocation of Foxo1 and Smad in hepatocytes. (A-D) Eight-week-old male wild-type C57BL/6J mice received a 3-hour acute (ferrous sulfate, 2 mg/kg body weight, oral gavage) or 3-day chronic iron challenge (rodent diet containing 8.24 g carbonyl iron per kg). (A-C) Serum iron levels (A), Bmp6 mRNA (B), and Hamp1 mRNA (C) were measured in the liver tissues. (D) Nuclear Foxo1 and Smad proteins were detected (left) and quantified (right) in the hepatocytes. (E-F) Primary hepatocytes treated with holo-Tf or BMP6 at indicated time duration. (E-F) Foxo1 and Smad proteins were measured by nucleocytoplasmic separation (E) and quantified (F). (G) Primary hepatocytes treated with holo-Tf or/and BMP6 for 7 hours; Foxo1 and Smad proteins were measured by nucleocytoplasmic separation (left panel) and quantified (right panel). (H) Hepcidin (Hamp1) mRNA expression levels in the primary hepatocytes treated with holo-Tf and/or BMP6 at indicated time duration. (I-K) Primary hepatocytes treated with holo-Tf and/or BMP6 for 7 hours; the immunofluorescence (I), corresponding 3-dimensional visualization (J), percentage (K) (left panel) and mean fluorescence intensity (MFI, right panel) of nuclear Foxo1 and Smad1 were analyzed. Nuclear translocation and MFI of Foxo1 and Smad1 were quantified using 10 representative images from 3 independent experiments (scale bar, 20 μm). Primary hepatocytes in panels E-K were isolated from 8-week-old male wild-type 129S2/SvPasCrl mice. The concentration of holo-Tf was 30 μM and BMP6 was 20 ng/mL. Results from quantitative polymerase chain reaction (qPCR) were obtained from 3 biological replicates. Quantification of western blot was obtained from 3 independent experiments. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. The P value was calculated by the 1-way analysis of variance (ANOVA) with the Sidak multiple comparison test. For panels A-D, significance label indicates the comparison between treated group and untreated group. For panels F,H, significance label indicates the comparison with the 0-time point, a negative control cultured for 24 hours without holo-Tf or BMP6 treatment.

Iron induces the nuclear translocation of Foxo1 and Smad in hepatocytes. (A-D) Eight-week-old male wild-type C57BL/6J mice received a 3-hour acute (ferrous sulfate, 2 mg/kg body weight, oral gavage) or 3-day chronic iron challenge (rodent diet containing 8.24 g carbonyl iron per kg). (A-C) Serum iron levels (A), Bmp6 mRNA (B), and Hamp1 mRNA (C) were measured in the liver tissues. (D) Nuclear Foxo1 and Smad proteins were detected (left) and quantified (right) in the hepatocytes. (E-F) Primary hepatocytes treated with holo-Tf or BMP6 at indicated time duration. (E-F) Foxo1 and Smad proteins were measured by nucleocytoplasmic separation (E) and quantified (F). (G) Primary hepatocytes treated with holo-Tf or/and BMP6 for 7 hours; Foxo1 and Smad proteins were measured by nucleocytoplasmic separation (left panel) and quantified (right panel). (H) Hepcidin (Hamp1) mRNA expression levels in the primary hepatocytes treated with holo-Tf and/or BMP6 at indicated time duration. (I-K) Primary hepatocytes treated with holo-Tf and/or BMP6 for 7 hours; the immunofluorescence (I), corresponding 3-dimensional visualization (J), percentage (K) (left panel) and mean fluorescence intensity (MFI, right panel) of nuclear Foxo1 and Smad1 were analyzed. Nuclear translocation and MFI of Foxo1 and Smad1 were quantified using 10 representative images from 3 independent experiments (scale bar, 20 μm). Primary hepatocytes in panels E-K were isolated from 8-week-old male wild-type 129S2/SvPasCrl mice. The concentration of holo-Tf was 30 μM and BMP6 was 20 ng/mL. Results from quantitative polymerase chain reaction (qPCR) were obtained from 3 biological replicates. Quantification of western blot was obtained from 3 independent experiments. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. The P value was calculated by the 1-way analysis of variance (ANOVA) with the Sidak multiple comparison test. For panels A-D, significance label indicates the comparison between treated group and untreated group. For panels F,H, significance label indicates the comparison with the 0-time point, a negative control cultured for 24 hours without holo-Tf or BMP6 treatment.

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