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

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HEP_24010_sm_suppFig-S1.tif5897KSupplementary Figure 1. Effects of chaetocin on tumor growth and hypoxic responses in MEF-driven fibrosarcomas. A) Representative tumor images of HIF-1α(+/+) and (-/-) fibrosarcomas grown in mice. After tumors grew to reach a volume of 100-150mm3, mice were treated with DMSO or chaetocin (0.25 mg/kg) once a day for two weeks. Mice and tumors were photographed on the day after the last injection. B) Tumor tissues were fixed by 4% paraformaldehyde, paraffin-embedded, and cut to 6 μm sections. The slides were stained with hematoxylin and eosin (1st row), or immunostained with anti-HIF-1α (2<nd</ row) or anti-CD31 (3rd row). The immunostained sections were visualized by the avidin-biotin-horseradish peroxidase method using diaminobenzidine as the chromogen, and lightly counterstained with hematoxylin. TUNEL staining (4th row) was performed to detect apoptotic cells in the sepcimens. C) Twelve mice (three sets, four per set) were intraperitoneally injected with DMSO or 0.25 mg/kg of chaetocin for 1 week, and then exposed to room air (N) or 10% O2/90% N2 (H) at 1 atmosphere in an air-tight plastic chamber, which was humidified and ventilated to minimize pCO2 changes. Kidneys were removed after 8 hour incubation in the chamber, and then erythropoietin (EPO) and η-actin mRNAs were analyzed by a semi-quantitative RT-PCR method.
HEP_24010_sm_suppFig-S2.tif329KSupplementary Figure 2. Effect of chaetocin on the hypoxic induction of HIF-1α in non-hepatic cancer cell lines. HCT116 colon, MCF7 breast and A549 lung cancer cells were pretreated with chaetocin (Ch) at indicated concentrations 1 hour prior to normoxic or hypoxic incubation for 8 hours.
HEP_24010_sm_suppFig-S3.tif218KSupplementary Figure 3. Effects of chaetocin on viabilities of hepatoma cells. A) Cell viability was measured by the MTT-labeling method. Hep3B and HepG2 cells were treated with chaetocin for 24 hours (left) or 48 hours (right) under normoxia or moderate hypoxia (1% O2), and then 100 μl of MTT (5 mg/ml) were added into 1 ml of medium and cells were further incubated for 3 hours. The reduced MTT levels were measured at 570 nm. B) Hep3B and HepG2 cells were treated with chaetocin for 48 hours under normoxia or severe hypoxia (0.1% O2) conditions, and cell viabilities were measured by using MTT dye. The arrows indicate the concentration range of chaetocin inhibiting HIF-1α. Each point represents the mean and SD of four independent experiments. Data were statistically analyzed using the two-sided Mann-Whitney U test, and * or # denotes p<0.05 vs. the corresponding value of normoxic Hep3B or HepG2, respectively.
HEP_24010_sm_suppFig-S4.tif232KSupplementary Figure 4. Effects of chaetocin on HIF-1-mediated hypoxic responses. A) Hep3B cells were co-transfected with 1 μg each of the VEGF-promoter reporter gene and the β-gal gene. Cells were treated with chaetocin in normoxia or hypoxia for 16 hours, and luciferase activities (n=4) were measured by a luminometer. B-D) Effect of chaetocin on the glycolytic ATP production. Hep3B cells were treated with chaetocin (100 nM) 1 hour prior to incubate normoxic or hypoxic conditions for 36 hours. Intracellular ATP levels (B) were measured using a bioluminescence method. Pyruvate (C) and lactate (D) levels were analyzed as described in Supporting Materials and Methods. Each bar represents mean+SD, and * denotes p<0.05 vs. the hypoxic control by the two-sided Mann-Whitney U test.
HEP_24010_sm_suppFig-S5.tif817KSupplementary Figure 5. Verification of purchased chaetocin. Hep3B cells were co-transfected with EPO-enhancer reporter and β-gal plasmids. Hep3B cells were treated with chaetocin purchased from three different companies, and then incubated under normoxic or hypoxic conditions for 16 hours. Each bar represents the mean and SD, and * denotes p<0.05 vs. the hypoxic control by the two-sided Mann-Whitney U test. D) Hep3B cells were treated for 8 hours with chaetocin (50 or 100 nM) purchased from three different companies, and proteins were measured by immunoblotting.
HEP_24010_sm_suppFig-S6.tif437KSupplementary Figure 6. Effect of chaetocin on HIF-1α expression. A) Hep3B cells were treated with 130 μM desferrioxamin (DFO) in the presence or absence of 100 nM chaetocin for 8 hours. HIF-1α protein was detected by immunoblotting. B) VHL-null RCC4 and p53-null HCT116 cells were incubated under normoxic and hypoxic conditions in the presence or absence of 100 nM chaetocin for 8 hours. HIF-1α protein levels were detected by immunoblotting. C) Hep3B cells were incubated under normoxic or hypoxic conditions for 4 hours in the presence or absence of 100 nM chaetocin, 25 μM LY294002 or 1 μM rapamycin 1 hour prior to normoxic or hypoxic incubation. The levels of phospho-Akt, total Akt, phospho-mTOR and total mTOR were detected by immunoblotting.
HEP_24010_sm_suppFig-S7.tif239KSupplementary Figure 7. Effect of chetomin on HIF-1α expression. A) Hep3B cells were co-transfected with EPO-enhancer reporter and β-gal plasmids. The cells were treated with chaetocin or chetomin for 1 hour and then incubated under normoxic or hypoxic conditions for 16 hours. Luciferase activities (means + SDs, n=4) were analyzed by a luminometer. B) Hep3B cells were treated with chaetocin or chetomin in hypoxia for 8 hours. HIF-1α and α-tubulin were analyzed by immunoblotting. C) Hep3B cells were treated with 100 nM of chaetocin or chetomin for 8 hours, and RT-PCR was performed to detect spliced and unspliced HIF-1αmRNA (Exon 11-Exon 12).
HEP_24010_sm_suppFig-S8.tif2090KSupplementary Figure 8. Representative images of Huh7 xenografts in mice. After Hur7 tumors grew to reach a volume of 100-150mm3, mice were treated with DMSO, chaetocin (0.25 mg/kg), doxorubicin (1 mg/kg), or chaetocin (0.25 mg/kg) plus doxorubicin (1 mg/kg) once a day for 10 days. Mice and tumors were photographed on the day after the last injection.
HEP_24010_sm_suppTab1.doc35KSupporting Table 1. Nucleotide sequences of primers used in experiments
HEP_24010_sm_suppinfo.doc39KSupporting Information.

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