Additional Supporting Information may be found in the online version of this article.

hep26490-sup-0001-suppinfo.doc71KSupporting Information
hep26490-sup-0002-suppfig1.tif2220KSupplementary Figure S1. Genistein treatment reduces the liver size in zebrafish embryos. Transgenic zebrafish expressing green fluorescent protein (GFP) under the control of the liver fatty acid protein (LFABP) promoter, Tg(LFABP:gfp) exhibit fluorescent green livers and were used to study genistein's effect on liver development. The Tg(LFABP:gfp) embryos were treated with genistein 7 μM (B, D) or vehicle only (A, C) starting from 26 hours post-fertilization through the time of observation. Photomicrographs of representative embryos in lateral views were obtained at 60 hours (A, B) and 80 hours (C, D) post-fertilization. The white arrowhead indicates the location of the liver. Scale bar represents 500 microns. N=12 embryos per group.
hep26490-sup-0003-suppfig2.tif2308KSupplementary Figure S2. Whole mount in situ hybridization for the endodermal marker FOXA3. Zebrafish embryos were treated with DMSO 1% (A) or genistein 7 μM (B) from 26-52 hours post-fertilization followed by fixation and in situ hybridization. Representative photomicrographs are shown (dorsolateral views). After treatment with genistein, FOXA3 expression (black arrowhead) is still present, but in a smaller area, consistent with a restriction in endodermal development following genistein treatment. Scale bar represents 100 microns. N=12 embryos per group.
hep26490-sup-0003-suppfig3.tif2722KSupplementary Figure S3. Genistein increases hepcidin expression relative to a liver specific marker and in isolated hepatocytes. A. Quantitative realtime RT-PCR for total hepcidin transcript levels relative to liver fatty acid binding protein (LFABP) were measured in pools of zebrafish embryos following treatment with DMSO 1% or genistein 7 μM from 26-52 hours post-fertilization. Following genistein treatment, hepcidin transcript levels were significantly increased relative to transcript levels of the liver-specific marker, liver fatty acid protein (LFABP) (8.93+2.46 vs. 1.75+0.64, p<0.05). N=3 pools of 20 embryos each. * denotes p=0.018. B. Quantitative realtime RT-PCR indicating an increase in hepcidin expression relative to β-actin in hepatocytes isolated from genistein-treated zebrafish embryos compared to the expression in hepatocytes isolated from DMSO-treated zebrafish embryos (40.5 vs 28.9). 58,000 hepatocytes were sorted by flow cytometry from each pool of 260 Tg(LFABP:GFP) zebrafish embryos at 72 hours post-fertilization following treatment with either DMSO 1% or genistein 7 μM from 28-72 hours post-fertilization. The hepcidin to β-actin expression is given as fold change over the expression in unsorted cells from DMSO-treated embryos. We observed similar results when isolating genistein-treated hepatocytes at 55 hours post-fertilization (data not shown).
hep26490-sup-0005-suppfig4.tif3279KSupplementary Figure S4. Genistein treatment does not cause anemia in zebrafish embryos. Following treatment with either DMSO 1% (A) or genistein 7 μM (B) from 26-52 hours post-fertilization, embryos were stained with o-dianisidine, which produces a red brown stain in tissues where hemoglobin is present. Photomicrographs of representative embryos in anterior views are shown. The red arrowheads indicate areas of hemoglobin staining overlying the developing heart. Scale bar indicates 100 microns. N=50 embryos per group.
hep26490-sup-0006-suppfig5.tif1278KSupplementary Figure S5. Genistein treatment does not alter ferritin levels in HepG2 cells. 2-5x106 HepG2 cells were plated in each well of a 6-well tissue-culture plate. After 24 hours, the cells were shifted to low serum medium (α-MEM/1% FBS) for 8 hours followed by treatment with 10 μM genistein or vehicle only (1% DMSO). After 24 hours of treatment, the cells were lysed. Cellular protein concentration was determined using the BCA Protein Assay. Cellular ferritin content was measured in triplicate assays using the Human Ferritin ELISA Kit #1810 (Alpha Diagnostic International, San Antonio, TX) The data are reported as micrograms of ferritin per microgram of protein. N=6 biological replicates for each condition.
hep26490-sup-0007-supptables.doc179KSupplemental Table S1. Sequences of primers and probes used in realtime quantitative PCR assays

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