Hepatocyte-specific Foxo1 overexpression alleviates iron overload in Hfe−/− mice. (A) A scheme to illustrate the validation of specificity of pLIVE-Foxo1-AAA in vivo. Four-week-old male Hfe−/− mice received a single tail vein injection of 200 μg pLIVE-Foxo1-AAA or an empty vector for 2 weeks (n = 3 mice in each group). (B-D) In the primary hepatocytes from Hfe−/− mice receiving 200 μg pLIVE-Foxo1-AAA or an empty vector for 2 weeks, Foxo1 mRNA (B), Hamp1 mRNA (C), Foxo1 protein, and pSmad1/5/8 (D) were measured. (E) The expression specificity of pLIVE-Foxo1-AAA was analyzed in the hepatocytes and nonhepatocytes from Hfe−/− mice receiving 200 μg pLIVE-Foxo1-AAA or an empty vector for 2 weeks (left). The nuclear Foxo1, Smad1, Smad4, and Smad5 in hepatocytes and nonhepatocytes were quantified (right). Quantification of western blot was obtained from 3 independent experiments. (F) A scheme to illustrate the validation of effectiveness in reducing iron overload in vivo. Four-week-old WT and Hfe−/− mice received the tail vein injection of 200 μg pLIVE-Foxo1-AAA or an empty vector at ages 4 and 5 weeks, respectively (2 times, 200 μg each time). Mice were euthanized at age 8 weeks (n = 6 in each group). (G-N) Iron-related phenotypes of WT and Hfe−/− mice received pLIVE-Foxo1-AAA or an empty vector treatment in panel F. (G-J) Liver nonheme iron (G), serum iron (H), transferrin saturation (I), and spleen nonheme iron levels (J) were measured. (K) Perls iron staining of the liver and spleen tissues. (L-N) Liver end point Foxo1 mRNA (L), Hamp1 mRNA (M), and serum hepcidin levels (N) were measured in WT and Hfe−/− mice receiving pLIVE-Foxo1-AAA or an empty vector in panel F. WT and Hfe−/− mice were C57BL/6J background. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. The P value was calculated by the Student t test for 2 group comparison or the 1-way ANOVA with the Sidak post hoc test for multiple comparisons in sex- and genotype-matched data. #P < .05; ##P < .01; ###P < .001. The P values were calculated by the 1-way ANOVA with the Sidak post hoc test for multiple comparisons between WT mice and Hfe−/− mice in sex-matched data.

Hepatocyte-specific Foxo1 overexpression alleviates iron overload in Hfe−/− mice. (A) A scheme to illustrate the validation of specificity of pLIVE-Foxo1-AAA in vivo. Four-week-old male Hfe−/− mice received a single tail vein injection of 200 μg pLIVE-Foxo1-AAA or an empty vector for 2 weeks (n = 3 mice in each group). (B-D) In the primary hepatocytes from Hfe−/− mice receiving 200 μg pLIVE-Foxo1-AAA or an empty vector for 2 weeks, Foxo1 mRNA (B), Hamp1 mRNA (C), Foxo1 protein, and pSmad1/5/8 (D) were measured. (E) The expression specificity of pLIVE-Foxo1-AAA was analyzed in the hepatocytes and nonhepatocytes from Hfe−/− mice receiving 200 μg pLIVE-Foxo1-AAA or an empty vector for 2 weeks (left). The nuclear Foxo1, Smad1, Smad4, and Smad5 in hepatocytes and nonhepatocytes were quantified (right). Quantification of western blot was obtained from 3 independent experiments. (F) A scheme to illustrate the validation of effectiveness in reducing iron overload in vivo. Four-week-old WT and Hfe−/− mice received the tail vein injection of 200 μg pLIVE-Foxo1-AAA or an empty vector at ages 4 and 5 weeks, respectively (2 times, 200 μg each time). Mice were euthanized at age 8 weeks (n = 6 in each group). (G-N) Iron-related phenotypes of WT and Hfe−/− mice received pLIVE-Foxo1-AAA or an empty vector treatment in panel F. (G-J) Liver nonheme iron (G), serum iron (H), transferrin saturation (I), and spleen nonheme iron levels (J) were measured. (K) Perls iron staining of the liver and spleen tissues. (L-N) Liver end point Foxo1 mRNA (L), Hamp1 mRNA (M), and serum hepcidin levels (N) were measured in WT and Hfe−/− mice receiving pLIVE-Foxo1-AAA or an empty vector in panel F. WT and Hfe−/− mice were C57BL/6J background. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001. The P value was calculated by the Student t test for 2 group comparison or the 1-way ANOVA with the Sidak post hoc test for multiple comparisons in sex- and genotype-matched data. #P < .05; ##P < .01; ###P < .001. The P values were calculated by the 1-way ANOVA with the Sidak post hoc test for multiple comparisons between WT mice and Hfe−/− mice in sex-matched data.

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