2.6.2. cells were washed three times with

2.6.2. Assessment of apoptotic cell death

 

HepG2 cells were seeded into 6-well culture
plates at a density of 5×105 cells per well
and incubated overnight at 37 °C in 5% CO2. After 24 h of
incubation, medium was discarded and
replaced with fresh serum free culture medium containing 2 µM of control CPG2 and
TAT-CPG2 (native and denatured forms) for 2 h. After 2 h of incubation, cells were washed three times
with PBS to remove non-transduced proteins then treated with 10 µM MTX and
incubated for 24 and 48 h. Apoptosis was assessed by using PE Annexin V
Apoptosis Detection Kit according to the manufacturer’s protocol.
Briefly, cells were harvested by trypsin and washed twice with the cold PBS buffer
and then resuspended in 1X binding buffer
at a concentration of 1×106 cells/ml. Then, 100 ?l of the solution (1 × 105 cells) was transferred
to a 5-ml culture tube. Five microliter of AnnexinV-FITC
and 5 ?l of 7-Amino-Actinomycin (7-AAD) were added to each tube. Cells were vortexed gently and incubated for 15 min at
room temperature in the dark. Finally, 400
?l of 1X binding buffer was added to each tube and analyzed by flow cytometer
(FACSCalibur, BD Biosciences).

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2.7. Western blot analysis and enzyme assay

For
western blot analysis, equal amount of each cell lysate was resolved by 12% sodium
dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The protein on
the gel was transferred onto a PVDF membrane and then blocked over-night with
1% skim milk in PBS at 4 ?C. The membrane was incubated with anti-His tag-peroxidase
antibody (1:500; Roche) for 1 h at 25 ?C. After three times washing the membrane
with the PBS containing 0.1% tween 20, and once with PBS without tween 20 (5 min for each), the
target protein was visualized with 3,3′-Diaminobenzidine (DAB) as the substrate.

Enzyme
activity was assayed by a modified method of McCullough et al. (McCullough et
al., 1971).
The reaction mixture containing 100 mM Tris-HCl at pH 7.3, 0.2 mM ZnSO4
and 50 µM MTX was equilibrated at 37 ?C for 10 min.  Decrease in the absorbance at 320 nm was
measured using Spectrophotometer (T80 UV/VIS
Spectrometer, PG Instruments). CPG2 activity was assessed using an
extinction coefficient of 8300 L mol-1 cm-1 for MTX. The
activity was calculated in units per milliliter (U/mL). One Unit is defined as
the amount of the enzyme required to catalyze the hydrolysis of 1
µmol MTX per minute at 37 ?C.

 

2.8. Quantification of intracellular
ROS production, GSH content and catalase (CAT) activity

 

The production of intracellular ROS was measured using
DCFH–DA (Jia et al., 2006). DCFH–DA passively enters the cell, where
it reacts with ROS to form a highly fluorescent compound, dichlorofluorescein
(DCF). HepG2 cells were seeded in a 6-well plate at 3 ×105 cells for
24 h.  Cells were treated with CPG2 and TAT-CPG2
for 2 h. Cells with or without TAT-CPG2 pre-treatment were
exposed to 10 µM MTX, and ROS detection was evaluated
at 12, 24 and 48 h after treatment. Following exposure, cells were trypsinized
and washed with the ice-cold PBS. Then, cells were co-incubated with serum-free
DMEM containing10 µM DCFH-DA for 30 min at 37 ?C in the dark. Subsequently, cells were harvested and
rinsed with the PBS buffer. Fluorescent intensities were measured using a
spectrofluorometer (FluoStar Omega, BMG labtech) at 485 nm excitation/530 nm
emission at the end of exposure times.

For determining GSH content and CAT activity, HepG2 cell
line was seeded in 6-well culture plates at 3 ×105 cells/well. Then,
cells were exposed to 10 µM MTX with or without pretreatment by TAT-CPG2 for
12, 24, and 48 h. Finally, cells were harvested and washed twice with PBS at 4 ?C.
Cells were then lysed in the cell lysis buffer (250 mM Tric-HCl, 10% v/v
glycerol, 1% triton X-100, 1 mM PMSF and 10 µg/ml leupeptin at pH 7.4) and centrifuged
at 10,000×g for 15 min. The supernatant was used for determination of the GSH
level and CAT activity.

GSH content was determined using the Ellman method (Ellman, 1959). For assay, 0.1 ml of the supernatant was added to 0.9
ml of 5%
trichloroacetic acid (TCA), and centrifuged at 2300 g for 15 min at room
temperature. Then, 0.5 ml of the supernatant was added into 1.5 ml of 0.01%
DTNB and the reaction was monitored at 412 nm using spectrophotometer. The GSH
level was expressed as ?g GSH/mg protein. CAT activity was determined based on
the decomposition of H2O2 (Aebi, 1984).  Decrease in the absorbance
at 240 nm was monitored for 2 min using spectrophotometer. Enzyme activity was
calculated using the extinction coefficient of H2O2 (43.6
mM?1 cm?1) and expressed as units/mg protein.

 

2.9. Statistical analysis