Flvcr1afl/fl;alb-cre mice showed the recombinant allele only in the liver ( Supplementary Figure 1B) and a strong reduction of hepatic Flvcr1a expression ( Supplementary Figure 1C and D). As expected, Flvcr1a mRNA could not be detected in primary hepatocytes isolated from GSK-3 activity Flvcr1afl/fl;alb-cre mice ( Supplementary Figure 1E), demonstrating that this mouse is a liver-specific knockout model

for Flvcr1a. Flvcr1afl/fl;alb-cre mice showed no gross liver abnormalities ( Supplementary Figure 1F). Blood analysis did not reveal any difference between Flvcr1afl/fl;alb-cre and Flvcr1afl/fl mice ( Supplementary Table 1). To evaluate if the deletion of Flvcr1a alters hepatic heme homeostasis, we analyzed the livers of 2- and 6-month-old Flvcr1afl/fl;alb-cre compared with those of an Flvcr1afl/fl counterpart. Hepatic heme and iron content were comparable at 2 months of age, but were Quizartinib molecular weight significantly higher in 6-month-old Flvcr1afl/fl;alb-cre than in Flvcr1afl/fl mice ( Figure 1A and B). Iron accumulation in 6-month-old Flvcr1afl/fl;alb-cre mice was further confirmed by Perl’s staining on liver sections ( Figure 1B). Consistently, Flvcr1afl/fl;alb-cre mice showed an enhanced HO activity as well as an increased bilirubin

excretion in the bile compared with Flvcr1afl/fl mice ( Figure 1C). In addition, Flvcr1afl/fl;alb-cre mice showed increased lipid peroxidation in the liver ( Figure 1D). The analysis of hepatic gene expression revealed that Flvcr1afl/fl;alb-cre mice up-regulated genes that encode for proteins involved in heme metabolism (Ho-1), 18 and 19 iron export (Fpn) 20 and 21 and storage (H- and L-Ferritin), 22 and antioxidant response (Txnrd1, γ-gcs, Sod1), 23 compared

with Flvcr1afl/fl mice ( Figure 1E and F; Supplementary Figure 2 for gene expression analysis of 2-month-old mice). On the other hand, expression of the other known heme exporter Abcg2 was not increased Vitamin B12 in the liver of Flvcr1afl/fl;alb-cre mice ( Supplementary Figure 3), indicating that no other heme exporter was able to compensate for the lack of Flvcr1a. The phenotype of liver-specific Flvcr1a knockout mice suggests that FLVCR1a-mediated heme export prevents hepatic heme accumulation. To further address this point, mice were injected with hemin, the substrate of FLVCR1a. One hour after heme injection, heme accumulated in the liver of both Flvcr1afl/fl;alb-cre and Flvcr1afl/fl mice at the same extent, but bilirubin production was significantly higher in Flvcr1afl/fl;alb-cre mice than in Flvcr1afl/fl mice, likely because of the enhanced HO activity ( Figure 2A and B). Consistently, if animals were pretreated with the heme analog Tin-Protoporphyrin IX that inhibits HO, before heme injection, Flvcr1afl/fl;alb-cre mice showed a significantly higher hepatic heme content 1 hour after heme infusion compared with control mice ( Figure 2C).