Fig. 6: Transferrin receptor restoration as well as Wnt pathway stimulation rescue iron homeostasis.
a, Western blot analysis of the protein levels of the transferrin receptor (TfR1) in intestinal organoids derived from aged mice and analyzed 40 days after transduction with a 3xFlag-Tfrc- expressing lentivirus. Mock (parental vector) was used as a negative control. Actin was used as a loading control. n = 3 mice per group were analyzed. b, Quantification of TET hydroxymethylase enzymatic activity in intestinal organoids as in a. n = 3 mice per group were analyzed. P value was calculated by a two-tailed Welch’s t-test. c, Bar chart showing the DNAm level of the Dkk2 gene promoter analyzed by BS pyrosequencing in intestinal organoids as in a. Parental organoids, untransduced organoids at day 0. n = 3 mice per group were analyzed. P value was calculated by a two-tailed Welch’s t-test. d, Representative images of the western blot analysis of the protein levels of TfR1 and FPN1 in intestinal organoids cultured in different media as indicated. ENRW (EGF, Noggin, R-spondin1, Wnt3a); ENRC (EGF, Noggin, R-spondin1, CHIR99021). GAPDH was used as a loading control. n = 3 mice per group were analyzed. For details, see the main text. e, Ferrous (Fe2+) iron levels in intestinal organoids as in d. n = 3 mice per group were analyzed. P value was calculated by one-way analysis of variance (ANOVA). f, Quantification of TET hydroxymethylase enzymatic activity in intestinal organoids as in d. n = 3 mice per group were analyzed. P value was calculated by one-way ANOVA. g, Representative images of the western blot analysis of the protein levels of the TfR1 and FPN1 in intestinal organoids treated with IFNγ (0.2 ng ml−1) or with both IFNγ (0.2 ng ml−1) and Wnt3a (100 ng ml−1) for 5 days as indicated. GAPDH was used as a loading control. n = 3 mice per group were analyzed. For details, see the main text. h, Ferrous (Fe2+) iron levels in intestinal organoids as in g. n = 3 mice per group were analyzed. P value was calculated by one-way ANOVA. i, Quantification of TET hydroxymethylase enzymatic activity in intestinal organoids as in g. n = 3 mice per group were analyzed. P value was calculated by ordinary one-way ANOVA. Error bars in bar charts represent the s.d.
