Supplemental Material
Animal experiments. Wild-type mice were maintained on a C57BL/6 background. GFP-LC3
transgenic mice (C57BL/6 background) were generated by Dr. N. Mizushima (Tokyo Medical
and Dental University, Japan) and were purchased from RIKEN (Japan). All animals received
humane care. All procedures were approved by the Institutional Animal Care and Use
Committee of the University of Kansas Medical Center. Mice were either given saline (n=5, i.p.)
or APAP (500 mg/kg) (n=6, i.p.). To modulate autophagy in vivo, wild type C57BL/6 mice were
injected (i.p.) with CQ (60 mg/kg) or rapamycin (2 mg/kg) or DMSO (2% DMSO in saline, 10
µL/g) immediately followed by APAP (500 mg/kg) injection. All mice were sacrificed 6 hrs
later. The serum from the blood samples was used for determination of alanine aminotransferase
(ALT) activities (Pointe Scientific, Canton, MI). Hematoxylin and eosin (HE) staining of liver
sections was performed as described previously.1 Total liver lysates were prepared using RIPA
buffer [1% NP40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl (lauryl) sulfate]. Cryo-liver
sectionings and fluorescence microscopy were performed as described before.2
Primary hepatocytes culture. As described previously,2 murine hepatocytes were isolated by a
retrograde, non-recirculating perfusion of livers with 0.05% Collagenase Type IV (Sigma). Cells
were cultured in William’s medium E with 10% fetal bovine serum but no other supplements for
2 hrs for attachment. Cells were then cultured in the same medium without serum overnight
before treatment. All cells were maintained in a 37°C incubator with 5% CO2.
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Antibodies. Antibodies used in the study were Tom20 and GFP (Santa Cruz Biotechnology),
p62 (Abnova), β-Actin (Sigma), GAPDH, total and phosphorylated 70-kDa ribosomal protein S6
kinase-1 (p70S6K), translational initiation factor 4E binding protein-1 (4EBP1) (Cell Signaling),
HMGB1 (Abcam), and cytochrome c (BD). The rabbit polyclonal anti-LC3 antibody was
described previously.3 HRP-conjugated and Cy3- conjugated secondary antibodies were from
Jackson ImmunoResearch.
Immunoblot assay. Cells were washed in PBS and lysed in RIPA buffer. Twenty micrograms
of protein from each sample were separated by SDS-PAGE and transferred to PVDF membranes.
The membranes were stained with primary antibodies followed by secondary horseradish
peroxidase-conjugated antibodies. The membranes were further developed with SuperSignal
West Pico chemiluminescent substrate (Pierce).
Electron Microscopy (EM). Cells were fixed with 2.5% glutaraldehyde in 0.1 mol/L phosphate
buffer (pH 7.4), followed by 1% OsO4. After dehydration, thin sections were stained with uranyl
acetate and lead citrate for observation under a JEM 1011CX electron microscope (JEOL).
Images were acquired digitally. The average number of autophagosomes from each cell was
determined from a randomly selected pool of 15 to 20 fields under each condition.
Microscopy for autophagy and cell death. To examine autophagy, primary hepatocytes were
seeded in a 12 well-plate (2  105 in each well) and infected with adenovirus-GFP-LC3 (100
viral particles per cell) overnight. Cells were treated with APAP in the presence or absence of 3MA (10 mM), NAC (10 mM) or CQ (20 µM). After treatment, cells were fixed with 4%
2
paraformaldehyde in phosphate buffered saline (PBS) for 2 hours at room temperature or kept at
4 C for microscopy. Fluorescence images were acquired under a Nikon Eclipse 200
fluorescence microscope with MetaMorph software. For the immunostaining assay, cells were
immunostained with the anti-HMGB1 antibody followed by Cy3-conjugated secondary antibody
as previously described.2 Cell death and morphological changes were analyzed with propidium
iodide (PI) staining (1 µg/mL) and phase-contrast imaging. All images were obtained using
digital fluorescence microscopy as described above.
Measurement of ROS production. Intracellular ROS was measured with the fluoroprobe 2′,7′dichlorofluorescein diacetate (DCFH-DA) as described previously.2 Briefly, primary hepatocytes
were treated with APAP (5 mM) in the presence or absence of CQ (20 µM) for 6 hrs. The cells
were further incubated with 2.5 μM DCFH-DA for 15 min at 37 °C followed by fluorescence
microscopy. Alternatively, the cells were stained with DCFH-DA (2.5 µM) for 15 min and
washed two times, and lysed in 0.2% Triton X 100 in PBS and equal amount of lysates were
transferred to a 96 well-plate and the DCF fluorescence intensity was measured using a plate
reader (Tecan, Durham, NC).
Statistical Analysis. Experimental data were subjected to Student t-test or one way analysis of
variance analysis with Scheffé’s post hoc test where appropriate. P<0.05 was considered
significant.
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Supplemental Figure 1. No obvious necrotic cell death is induced by various
concentrations of APAP after 6 hours treatment in primary mouse hepatocytes. (A) Primary
cultured mouse hepatocytes were treated with various concentrations of APAP (0, 1.25, 2.5, 5
and 10 mM) for 6 hrs followed by PI staining. PI positive cells with intact nuclei were quantified
for each experiment (n=3). More than 300 cells were counted in each individual experiment. .
(B) Primary mouse hepatocytes were treated with APAP (5 Mm) for 6 hrs and then processed for
EM analysis. Representative EM images of autophagosomes containing mitochondria in APAPtreated hepatocytes were shown.
Supplemental Figure 2. Suppression of autophagy enhances APAP-induced ROS
production. Primary cultured mouse hepatocytes were treated with APAP (5 mM) in the
absence or presence of CQ (20 µM) for 6 hrs. The cells were stained with DCFH-DA (2.5 µM)
for 15 min and washed two times with PBS followed by fluorescence microscopy.
Representative photographs were shown in (A). (B) Quantification of DCF fluorescence
intensity. Data are means ± SD from three independent experiments.
Supplemental Figure 3. APAP-induced necrosis in hepatocytes is suppressed by rapamycin.
(A) Representative overlayed images of phase-contrast with PI staining of primary mouse
hepatocytes treated for 24 hrs. Panel a: non-treated; panel b: TNF-α (10 ng/mL) plus ActD (0.1
µg/mL); panel c: APAP (10 mM). Panel d: enlarged photograph from the boxed area in panel c.
(B) PI positive cells were quantified for each experiment (n=3). More than 300 cells were
counted in each individual experiment *: p<0.01. (C) Representative EM images of control and
APAP-treated hepatocytes for 24 hrs were shown. N: nucleus; m: mitochondria. (D) Primary
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mouse hepatocytes were co-treated for 24 hrs as indicated: panel a: DMSO; panel b: APAP (10
mM)+ DMSO (4 µL/mL); panel c: APAP (10 mM) + Rap (2 µM); and panel d: Rap ( 2 µM).
Representative overlayed images of phase-contrast with PI staining were shown.
Supplemental Figure 4. Rapamycin does not affect the initial glutathione (GSH) depletion
induced by APAP. (A) Wild type C57BL/6 mice were injected (i.p.) with rapamycin (2 mg/kg)
or DMSO (2% DMSO, 10 µL/g) immediately followed by APAP (500 mg/kg) injection for 2
hrs. Blood ALT levels were quantified (n=3 mice). (B) The liver content of total GSH was
determined as described previously4 (C) Wild type C57BL/6 mice were injected (i.p.) with CQ
(60 mg/kg), rapamycin (2 mg/kg) or DMSO (2% DMSO, 10 µL/g) immediately followed by
APAP (500 mg/kg) injection for 6 hrs. The liver content of total GSH was then determined.
REFERENCES:
1. Ding WX, Li M, Chen X, Ni HM, Lin CW, Gao W, Lu B, Stolz DB, Clemens DL, Yin XM.
Autophagy reduces acute ethanol-induced hepatotoxicity and steatosis in mice. Gastroenterology
2010;139:1740-52.
2. Ding WX, Ni HM, DiFrancesca D, Stolz DB, Yin XM. Bid-dependent generation of oxygen
radicals promotes death receptor activation-induced apoptosis in murine hepatocytes. Hepatology
2004;40:403-13.
3. Ding WX, Ni HM, Gao W, Chen X, Kang JH, Stolz DB, Liu J, Yin XM. Oncogenic
transformation confers a selective susceptibility to the combined suppression of the proteasome
and autophagy. Mol Cancer Ther 2009;8:2036-45.
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4. Saito C, Lemasters JJ, Jaeschke H. c-Jun N-terminal kinase modulates oxidant stress and
peroxynitrite formation independent of inducible nitric oxide synthase in acetaminophen
hepatotoxicity. Toxicol Appl Pharmacol 2010;246:8-17.
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