We validated our hits with quantitative realtime RT-PCR assays for Hepcidin expression and characterized them

by their effects on genes regulated by BMP’s or Stat3, as well as Western blots to detect phosphorylation of Smad1,5,8 or Stat3. We confirmed 16 small molecule Hepcidin stimulating agents in a broad range of functional classes. All of the chemicals identified increased expression of bone morphogenic protein-dependent and/or Stat3-dependent genes, however none of them strongly increased phosphorylation of Smad1,5,8 Anti-infection Compound Library in vivo or Stat3. Several of the Hepcidin stimulatory chemicals inhibit growth factor receptor dependent signaling (AG1296, GTP 14564, AS252424, 10058-F, SU6668, and pterostilbene), decrease inflammation (leflunomide, amlexanox), or impair DNA repair and promote apoptosis (daunorubicin, 9-aminocridine, ethacridine), while the small molecules, vorinostat and SB 204741, inhibit histone deacetylase and serotonin receptor 2B, respectively. Two of the molecules, ipriflavone and vorinostat, were active at concentrations that were 10-fold below those required for genistein’s effect and thus appear to be intriguing candidates for further development. The human hepatocarcinoma cell line, HepG2, (American Type Culture Collection, Manassas, VA) was maintained in α-Minimum Essential Medium

(α-MEM)/10% certified endotoxin-free fetal bovine serum (FBS)/1% penicillin–streptomycin (Life Technologies, Grand Island, NY) at 37 °C, 5% CO2. To generate a Hepcidin reporter cell line, HepG2 cells, were transfected using SuperFect Alectinib mouse (Qiagen, Valencia, CA) transfection PAK6 reagent and a reporter construct including a 2.7 kb fragment of the

human Hepcidin promoter upstream of a firefly luciferase promoter (gift of Drs. Ganz and Nemeth). Transfected clones were selected for resistance to G418 (Life Technologies) and subsequently maintained in the conditions described above with the addition of G418 1 mg/ml. Bone morphogenic protein 6 (BMP6) (R&D Systems, Minneapolis, MN), interleukin-6 (R&D Systems), and genistein (Sigma-Aldrich, St. Louis, MO) were used as positive controls for Hepcidin-luciferase activity, while dorsomorphin (#171260, Calbiochem, Billerica, MA) was used as a negative control. WP1066 (#573097, Calbiochem) was used as an inhibitor of Stat3 signaling. Interleukin-6, EGF, FGF, PDGF, and VEGFA were obtained from R&D Systems. The Institute of Chemistry and Cell Biology (ICCB) Screening Facility at Harvard Medical School provided drug libraries. The complete list of chemicals screened and the screening data are provided in Supplementary Table 1. The day before the addition of compounds, HepG2-Hepcidin luciferase cells were plated at 5000 cells in 30 μl per well of a 384-well microtiter plate (Nunc 142762) in α-MEM/1% penicillin/streptomycin using a WellMate MicroPlate Dispenser (Thermo Scientific, Rockford, IL).