670
2.4. Liver histology.
Histological grading of hepatic necrosis was performed by two blinded
oervbsers using hematoxilin and eosin (H&E)-stained sections as follows: 30% of
the total area necrotic (1 point); 30–60% of the total area necrotic (2 points); 60%
of the total area necrotic (3 points).
2.5. Generation of immortalized hepatocyte cell lines.
The detailed protocol for the generation of immortalized neonatal
hepatocytes from wild-type and PTP1B
-/-
mice is described in (27). For re-
expression of PTP1B in deficient cells, PTP1B
-/-
neonatal hepatocytes were
reconstituted with retroviral Myc-tagged PTP1B (kindly provided by M. L.
Tremblay, McGill Cancer Center, Quebec, Canada) and four pools of infected cells
were selected with hygromycin B (200 μg/ml) for 2 weeks. As a control, PTP1B
-/-
were infected with an empty vector (pBabe hygro). The expression of PTP1B in the
different cell lines was assessed by Western blot.
2.6. Isolation and culture of primary mouse hepatocytes.
Mouse hepatocytes were isolated from male mice (8-12 weeks-old) by
perfusion with collagenase and cultured as described (27).
2.7. PTP1B immunohistochemistry.
PTP1B expression was analyzed by immunohistochemistry in human liver
samples as previously described (29).
2.8. Determination of Reactive Oxygen Species (ROS).
Cellular ROS were quantified by flow cytometry using the dichlorofluorescin
(DCFH) probe. APAP-stimulated cells were detached by trisinization and collected
by centrifugation at 2.500 x g for 4 min. Then, cells were washed and resuspended
in 500 microliters of PBS. DCFH (10 μM) and propidium iodide (0,002% w/v) were
added 10 min before measurement. Fluorescence was measured with 488-nm
laser excitation and 510-nm for emission.
2.9. Analysis of caspase-3 activity.
At the end of the treatments, cells were scraped off, collected by
centrifugation at 2.500 x g for 5 min and lysed at 4ºC in 5 mM Tris/HCl pH 8, 20
mM EDTA, 0,5% Triton X-100. Lysates were clarified by centrifugation at 13.000 x
g for 10 min. Reaction mixtures contained 25 microliters of cell lysate, 325
microliters of assay buffer (20 mM HEPES pH 7.5, 10% glycerol, 2 mM
dithiothreitol) and 20 µM caspase-3 substrate (Ac-DEVD-AMC). After 2-hour
incubation at 37ºC in the dark, enzymatic activity was measured in a luminescence
spectrophotometer (Perkin Elmer LS-50, Norwalk, CT) ( excitation, 380 nm;
emission, 440 nm). We define a unit of caspase-3 activity as the amount of active
enzyme necessary to produce an increase in 1 arbitrary unit in the fluorimeter
