Fig 2: Effects of Cp administration on iron accumulation and iron homeostasis-related proteins expression in the liver. (a) Iron evaluation by ICP-MS in the liver of mice after 2 months of treatment with an intraperitoneal injection of Cp 5 µg/g, administered every 5 days (KO+Cp; WT+Cp) or saline (KO, WT). (b) Hepcidin level measured by ELISA in the liver lysates of mice treated as in (a). (c) Western blot analysis of transferrin receptor 1 (TfR1), ferroportin 1 (Fpn), ferritin (Ft) and actin expression in the liver of animals after treatment with Cp (KO+Cp; WT+Cp) or saline (KO, WT). Cropped images are from the same or twin SDS-PAGE and Western blot but at a different exposure. (d–f) Densitometric analysis of Western blot signals for TfR1 (d), Fpn (e) and Ft (f), performed by ImageJ software; values are reported as optical density (OD) normalized for actin. Data: mean ± SEM of animal groups, each dot corresponds to one animal (CpKO, WT n = 5; CpKO+Cp, WT+Cp, n = 6); statistical p-values were evaluated by two-ways ANOVA and reported as value for overall ANOVA, value for Benjamini-Hochberg adjustment for multiple ANOVA tests related to liver features (adj-ANOVA), for genotype variable (gen), for treatment variable (treat) and for the interaction of genotype/treatment (gen/treat).

Fig 3: Deposition of metal ions and expression of iron homeostasis-related proteins in perigonadal adipose tissue. (a) Iron, copper, and zinc metal ions evaluation by ICP-MS in pgAT from mice at 10 months of age after 2 months of treatment with an intraperitoneal injection of Cp 5 µg/g, administered every 5 days (KO+Cp; WT+Cp) or saline (KO, WT). (b) Hepcidin level measured by ELISA in pgAT lysates of mice treated as in (a). (c) Western blot analysis of transferrin receptor 1 (TfR1), ferroportin 1 (Fpn), ferritin (Ft) and actin expression in pgAT of mice treated as in (a). Cropped images are from the same or twin SDS-PAGE and Western blot but at a different exposure. (d–f) Densitometric analysis of Western blot signals for TfR1(d), Fpn (e) and Ft (f) was performed with ImageJ software; values are reported as optical density (OD) normalized for actin. Data: mean ± SEM of animal groups; each dot corresponds to one animal (CpKO, WT n = 5; CpKO+Cp, WT+Cp, n = 6); statistical p-values were evaluated by two-ways ANOVA and reported as value for overall ANOVA, value for Benjamini-Hochberg adjustment for multiple ANOVA tests related to adipose tissue features (adj-ANOVA), for genotype variable (gen), for treatment variable (treat) and for the interaction of genotype/treatment (gen/treat).