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Supplementary material for this article can be found on the H EPATOLOGY website ( http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html ).

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suppfig1.tif1824KDAPI staining of hepatocytes and activated HSCs. Cells were treated for 3 hr with gliotoxin (concentration as indicated) or 20ng/ml TNFa / 10µM cycloheximide (TNFa/CHX) for 16 hr followed by DAPI staining as outlined in methods section. Data typical of at least 3 separate experiments.
suppfig2a.tif1117KGliotoxin metabolism in hepatocytes and HSCs. A, Hepatocytes and activated HSCs were prepared a cultured in 6 well plates as outlined in Materials and Methods section. Culture medium was removed and the cells were washed twice with 1.5 ml of HEPES/HBSS buffer. Hepatocytes were then incubated with 1.5ml of pre-warmed HEPES/HBSS buffer containing 50µM gliotoxin - hepatocytes (?) or activated HSCs (?). Cells were returned to an humidified incubator at 37oC and HEPES/HBSS medium samples were taken at various times and directly analyzed by HPLC. No change in gliotoxin concentration occured when gliotoxin was incubated in HEPES/HBSS buffer in culture plates that contained no cells (data not shown). Inset, effect of initial gliotoxin concentration on the accumulation of gliotoxin after 45 minutes incubation in hepatocytes (?) or activated HSCs (?), mean and standard deviation of 3 samples from the same experiment. Data typical of 3 separate experiments.
suppfig2b.tif2202KGliotoxin metabolism in hepatocytes and HSCs. B, HPLC chromatograms of 50µM gliotoxin in (i) HEPES/HPLC buffer; (ii) after incubation with hepatocytes for 45 minutes; (iii) 10µM gliotoxin incubation with activated HSCs for 120 minutes and iv) 50µM gliotoxin incubation with activated HSCs for 120 minutes.
suppfig2c.tif2202KGliotoxin metabolism in hepatocytes and HSCs. C. Timecourse for the levels of gliotoxin metabolites produced by hepatocytes incubated with 50µM gliotoxin. Concentrations of metabolites are determined assuming an identical extinction coefficient as gliotoxin at the detecting wavelength of 260nm. Data from one experiment typical of at least 4 separate experiments.
suppfig3a.tif994KEffect of gliotoxin on the levels of glutathione in a cell-free system, hepatocytes and activated HSCs. A. GSH (50µM) in HEPES/HBSS buffer was incubated with 50µM gliotoxin (+ GLIOTOXIN) or DMSO vehicle solvent (+ DMSO) at room temperature. At various times, samples were withdrawn and derivatized as outlined in the methods section and GSH and GSSG levels determined by HPLC. Data are expressed as equivalents of GSH (i.e. GSSG is equivalent to 2 times GSH); "gliotoxin conjugate?" not detected, determined after calculating recoveries.
suppfig3b.tif2202KEffect of gliotoxin on the levels of glutathione in a cell-free system, hepatocytes and activated HSCs. B. HPLC analysis of 250µM gliotoxin in HEPES/HBSS buffer incubated for 1 hour with i) no furthur addition; ii) 500µM DTT; iii) 500µM GSH µM or iv) 500µM PDTC. Chromatograms for v) DTT alone; vi) GSH alone or vii) PDTC alone. Chromatograms are for the same injection volume and are presented on identical Y axis (absorbance) scales.
suppfig3c.tif2202KEffect of gliotoxin on the levels of glutathione in a cell-free system, hepatocytes and activated HSCs. C. Timecourse for changes in glutathione levels in hepatocytes treated with 50µM gliotoxin. Hepatocytes were treated with gliotoxin and GSH levels determined after derivatization as outlined in methods section. Data are the mean and standard deviation of 3 determinations from the same experiment typical of 3 separate experiments.
suppfig3d.tif2202KEffect of gliotoxin on the levels of glutathione in a cell-free system, hepatocytes and activated HSCs. D. Timecourse for changes in glutathione levels in activated HSCs treated with control vehicle solvent (¦), 1.5µM gliotoxin (?) or 50µM gliotoxin (?). GSH levels were determined after derivatization as outlined in methods section. Note that GSSG was not detected in HSCs. Data are the mean and standard deviation of 3 determinations from the same experiment typical of 3 separate experiments.
suppfig3e.tif984KEffect of gliotoxin on the levels of glutathione in a cell-free system, hepatocytes and activated HSCs. E. Levels of glutathione in hepatocytes and activated HSCs after treatment with the indicated concentration of gliotoxin (GT) or with 20ng/ml TNFa + 10µM cycloheximide (TNFa/CHX), 100nM microcystin (MC), 200µM chlorpromazine (CLPRZ), 1mM N-ethylmaleimide (NEM) (all for 3 hours except TNFa/CHX - for 16 hours). Control hepatocytes were found to contain 20 ± 1.5 nmole GSH per mg protein and 1 ± 0.1 nmole GSSG per mg protein. Control activated HSCs were found to contain 12 ± 1. 5 nmoles GSH per mg protein. Data are the mean and standard deviation of at least 3 separate determinations. *Significantly different level of GSH or GSSG versus equivalent control treated cells, P> 95% using Student's t test (two tailed).
suppfig4.tif2203KMechanism of toxicity of gliotoxin in rat hepatocytes. Inset, structure of gliotoxin and abbreviated structure showing the dithiol bridge.
suppfig5.tif2202KPathways of gliotoxin mediated apoptosis. This figure is a simplification of the pathways involved in TNFa mediated apoptosis in sensitive cells.

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