1. Gliotoxin-mediated apoptosis of activated human hepatic stellate cells
Young-Oh Kweon, Yong-Han Paik, Bernd Schnabl, Ting Qian, John J Lemasters, David A Brenner 
Journal of Hepatology 
Volume 39, Issue 1, Pages (July 2003)
DOI: /S (03)

2. Fig. 1
Gliotoxin induces cell death in activated HSCs. HSCs were treated with gliotoxin or TNF-α (30 ng/ml) for 1 h. Morphological changes were observed by phase-contrast microscopy (A). A time course and dose–response curve for the effect of gliotoxin on HSCs is shown. HSCs were treated with gliotoxin (100nM–32.5 μM). Cell viability was assessed after 1, 2, 4 and 8 h by trypan blue exclusion test. Data are shown as average percent viability ±SEM of 2–4 different experiments (B). HSCs were treated with gliotoxin (32.5 μM) and the cells were observed by confocal microscopy with propidium iodide staining (C). Primary mouse hepatocytes were treated with different concentrations of gliotoxin and cell viability was assessed after 1, 2, 4 and 8 h by trypan blue exclusion test. Data are shown as average percent viability ±SEM of 2–4 different experiments (D).
Journal of Hepatology  , 38-46DOI: ( /S (03) )

3. Fig. 2
Formation of ROS after gliotoxin treatment. HSCs plated in 6-well dishes on 40mm-diameter glass cover slips were incubated with buffer containing 10 μM DCFH-DA for 20 min at 37°C. Afterwards the formation of ROS was determined by fluorometric analysis (A) or confocal microscopic imaging analysis (B), HSCs were pretreated with or without NAC (5 mM) and then incubated with gliotoxin (1.5 μM). Phase contrast images were taken (C). Cell viability was assessed after incubation with different concentrations of NAC (D) and DTT (E) (1.5 μM of gliotoxin, 6 h incubation).
Journal of Hepatology  , 38-46DOI: ( /S (03) )

4. Fig. 3
ATP levels decrease at high doses of gliotoxin. HSCs were incubated with gliotoxin at different doses (0.3–32.5 μM) and ATP was measured using a luciferin/luciferase assay.
Journal of Hepatology  , 38-46DOI: ( /S (03) )

5. Fig. 4
Gliotoxin inhibits DNA binding activity of NFκB. HSCs were incubated with gliotoxin for 1 h. Mobility shift assay using 10 μg of nuclear protein extracts and a radiolabeled consensus NFκB site as probe was performed. Reactions were fractionated on a native 5% polyacrylamide gel.
Journal of Hepatology  , 38-46DOI: ( /S (03) )

6. Fig. 5
Gliotoxin induces mitochondrial depolarization, while untreated HSCs do not undergo mitochondrial depolarization. HSCs were loaded with red-fluorescing TMRM and green-fluorescing Mitotracker-green. After loading, HSCs were treated with gliotoxin (1.5 μM). Red and green fluorescences were monitored simultaneously in living cells by confocal microscopy. Images are shown at 10, 20, 30, 40 and 50 min after gliotoxin treatment (A). Images of untreated control cells are shown at 20, 40 and 60 min after gliotoxin treatment (B).
Journal of Hepatology  , 38-46DOI: ( /S (03) )

7. Fig. 6
Gliotoxin induces Annexin-V positive staining. HSCs were treated with gliotoxin (300nM) and stained using a FITC-conjugated Annexin-V antibody. The nucleus was stained with propidium iodide at the same time and the respective phase contrast images are shown (A). HSCs were treated with gliotoxin, fixed and subsequently stained with propidium iodide. Apoptotic cells lift off from the monolayer, therefore to focus on these the monolayer would fall below the plane of focus (B). Respective phase contrast microscopic images are shown below, most of the cells are rounded up and lift off from the surface monolayer (C).
Journal of Hepatology  , 38-46DOI: ( /S (03) )

8. Fig. 7
The mitochondrial depolarization is accompanied by cytochrome c release and caspase-3 activation. HSCs were treated with gliotoxin (1.5 μM) and S-100 fractions were prepared at the indicated time points and analyzed for cytochrome c content by Western blotting (A). HSCs were treated with gliotoxin (1.5 μM), lysed and then assayed for caspase-3 activity at 1-h intervals. Data are presented as average picomoles of AFC released per microgram of protein±SEM (B). HSCs were treated with gliotoxin (1.5 μM) alone or in combination with DEVD-cho (50 μM). Viability was assessed at the indicated time points. Average percent viability±SEM is shown (C).
Journal of Hepatology  , 38-46DOI: ( /S (03) )

9. Fig. 8
The combination of CsA and TFZ blocks the mitochondrial depolarization and release of cytochrome c but, not apoptosis of HSCs. HSCs were loaded with red-fluorescing TMRM and green-fluorescing Mitotracker-green. HSCs were then pretreated with CsA (5 μM) plus TFZ (12.5 μM) and treated with gliotoxin (1.5 μM). Red fluorescence and green fluorescence were monitored simultaneously in living cells by confocal microscopy. Red color indicates pixel saturation. Images are shown at 20, 40, 80 min after gliotoxin treatment (A). HSCs were treated with CsA (5 μM) and TFZ (12.5 μM) before treatment with gliotoxin (1.5 μM). Cell viability was assessed by trypan blue exclusion test. Average percent viability±SEM is shown (B). HSCs were pretreated with CsN plus TFZ and were treated with gliotoxin (1.5 μM) or vehicle for 1 h. S-100 fractions from treated and control cells were prepared and analyzed for cytochrome c content by Western blotting (C). HSCs were treated with gliotoxin (1.5 μM), lysed and then assayed for caspase-3 activity at 1-h intervals. Data are presented as average picomoles of AFC released per microgram of protein per hour±SEM (D). HSCs were pretreated with CsA (5 μM) plus TFZ (12.5 μM) and then treated with gliotoxin (1.5 μM). Cells were stained using a FITC-conjugated Annexin-V antibody and propidium iodide (E). HSCs were pretreated with CsA (5 μM) plus TFZ (12.5 μM) and then treated with gliotoxin (1.5 μM). Cells were stained with propidium iodide (F).
Journal of Hepatology  , 38-46DOI: ( /S (03) )