All experiments were approved by the animal ethical committee of the University of Torino (Italy). Heme content in tissues and bile was quantified by the oxalic acid method. Tissue nonheme iron content was determined by a colorimetric method using 4,7-diphenyl-1, INCB018424 purchase 10-phenantroline disulfonic acid (Sigma, St Louis, MO) as chromogen. HO activity was measured by spectrophotometric determination of bilirubin produced from hemin added as substrate. Lipid peroxidation from tissue extracts was measured using the colorimetric assay kit Bioxytech LPO-586 from Oxis International (Portland, OR). Total RNA
was extracted using Pure Link RNA Mini Kit (Ambion, Life Technologies Italia, Torino, Italy). One microgram total RNA was reverse transcribed using M-MLV reverse transcriptase and random primers (Life Technologies Italia). Quantitative real-time polymerase chain reaction was performed on a 7300 Real Time PCR System (Applied Biosystems, Life Technologies Italia). Primers and probes were designed using the ProbeFinder Venetoclax software (www.roche-applied-science.com). Tissue and cell proteins
were extracted as reported previously17 and concentration was determined using the Bio-Rad protein assay system (Bio-Rad, Munich, Germany). Fifty micrograms total protein extracts were separated on 8%−12% sodium dodecyl sulfate polyacrylamide gel electrophoresis and analyzed by Western blotting using antibodies against HO-1 (Stressgen, Victoria, Canada), L- and H-ferritin (kindly provided by Sonia Levi), ferroportin (Fpn; Alpha
Diagnostic Intl. Inc, San Antonio, TX), CYP1A1, CYP3A, CYP2E1, and actin (Santa Cruz Biotechnology, Inc., Dallas, TX). Tissues were fixed in 10% formalin overnight at room temperature and embedded in paraffin. Microtome sections, 5-μm thick, were stained with Perl’s reaction followed by methanol 3,3-diaminobenzidine (Boehringer Mannheim, Germany) development. ALAS activity was assayed by measuring ALA formation in liver homogenates after glycine addition. CYP1A1 activity was assessed by measuring ethoxyresorufin-O-deethylase activity in liver microsomes using 7-ethoxyresorufin as a substrate. CYP3A activity was assessed by measuring conversion of the substrate proluciferin-PFBE to luciferin (V8901 P450-Glo CYP3A4 Assay; Promega, Madison, WI). CYP2E1 Sclareol activity was determined by assaying the hydroxylation of p-nitrophenol to 4-nitrocatechol in the liver microsomal fraction. Results were expressed as mean ± SEM. Comparisons between 2 groups were performed with 2-sided Welch t tests and among more than 2 groups with 1- or 2-way analysis of variance followed by Bonferroni post test. P values <.05 were regarded as significant (∗P <. 05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001). See also Supplementary Material. To study the function of the heme exporter FLVCR1a in the liver, we generated a liver-specific Flvcr1a knockout mouse ( Supplementary Figure 1A).