HepG2 were prepared for western blot and enzyme

HepG2 cells were cultured in Dulbecco’s modified Eagle’s medium
containing 20 mM HEPES/NaOH (pH 7.4), 5 mM NaHCO3, 10% fetal bovine
serum, and antibiotics (100 ?g/ml streptomycin, 100 U/ml penicillin) at 37 °C
under humidified conditions of 95% air and 5% CO2. To evaluate
concentration and time dependency of TAT-CPG2 transduction, treatments were
carried out as follows: HepG2 cells were grown to
confluence on a six well plate and incubated with different
concentrations (0.5-4 ?M) of native
and denatured proteins for 2 h or with 2 ?M of native and denatured
proteins for various periods of time (15–120 min). Cells were incubated with 2
?M CPG2 for 2 h as the control group. To examine the intracellular stability of TAT-CPG2 protein, cells were treated with 2 µM TAT-CPG2 for 2 h.  Cells were then washed with PBS to remove non-transduced TAT-CPG2. Cells were further incubated in fresh culture medium for
2, 6, 12, 24, 36 and 48 h. After the above treatments, cells were treated
with trypsin-EDTA and washed with PBS. Cells were lysed with the lysis buffer (250 mM Tris-HCl, 10% v/v glycerol, 1% triton X-100, 1 mM PMSF, and 10 µg/ml leupeptin at pH 7.4).
After centrifugation at 15,000 × g for 30 min at 4
°C, cell extracts were prepared
for western blot and enzyme assay analyses.



2.6. Inhibitory effect of TAT-CPG2 against MTX- induced
toxicity in HepG2 cells

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2.6.1. Cell viability assay


Cell viability was evaluated using the MTT assay (Mosmann, 1983). In brief, HepG2
cells were seeded in 96-well plates in
a volume of 100 µl at a density of 1.0 × 104 cells/well. After 24 h
of incubation for growing and adherence, the medium was
replaced with the fresh medium containing various concentrations of
CPG2 and TAT-CPG2 for 2 h. After
treatment, culture medium was removed and cells were washed twice with ice-cold
PBS. Cells with or without TAT-CPG2 pretreatment were exposed
to different concentrations of MTX (0.1-100 µM) and incubated for 24 and 48 h.
In addition, effect of TAT-CPG2 on the cell viability was carried out at
concentrations of 0.5 to 4 µM. To examine the inhibitory effect of different protein concentrations against MTX-induced cell death,
HepG2 cells were treated with various concentrations of TAT-CPG2 for 2 h and
then exposed to 10 µM MTX for 48 h. Following all above treatments, cells were incubated
with MTT (0.5 mg/ ml) at 37 ?C for 4 h. The
medium was then removed and 100 µl of DMSO was added into each well and mixed
thoroughly on a shaker for 15 min to dissolve formazan crystals.
The absorbance was measured at 570 nm using a Microplate
Spectrophotometer (BioTek PowerWave XS2, USA). Cell viability was
calculated as a percentage of absorbance of sample compared to that of the


2.6.2. Assessment of apoptotic cell


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).


2.7. Western blot analysis and enzyme assay

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.

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
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


All experiments were performed three times independently. All data were reported as
the mean ± standard deviation (SD). Statistical significance was determined by
one-way and two-way analysis of variance (ANOVA) by Grappad prism 7 software. p
< 0.05, p < 0.01 and p < 0.001 considered to be significant.