2.6.2. cells were washed three times with
2.6.2. Assessment of apoptotic cell death HepG2 cells were seeded into 6-well cultureplates at a density of 5×105 cells per welland incubated overnight at 37 °C in 5% CO2. After 24 h ofincubation, medium was discarded andreplaced with fresh serum free culture medium containing 2 µM of control CPG2 andTAT-CPG2 (native and denatured forms) for 2 h. After 2 h of incubation, cells were washed three timeswith PBS to remove non-transduced proteins then treated with 10 µM MTX andincubated for 24 and 48 h. Apoptosis was assessed by using PE Annexin VApoptosis Detection Kit according to the manufacturer’s protocol.Briefly, cells were harvested by trypsin and washed twice with the cold PBS bufferand then resuspended in 1X binding bufferat a concentration of 1×106 cells/ml.
Then, 100 ?l of the solution (1 × 105 cells) was transferredto a 5-ml culture tube. Five microliter of AnnexinV-FITCand 5 ?l of 7-Amino-Actinomycin (7-AAD) were added to each tube. Cells were vortexed gently and incubated for 15 min atroom 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.
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7. Western blot analysis and enzyme assayForwestern blot analysis, equal amount of each cell lysate was resolved by 12% sodiumdodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The protein onthe gel was transferred onto a PVDF membrane and then blocked over-night with1% skim milk in PBS at 4 ?C. The membrane was incubated with anti-His tag-peroxidaseantibody (1:500; Roche) for 1 h at 25 ?C. After three times washing the membranewith the PBS containing 0.1% tween 20, and once with PBS without tween 20 (5 min for each), thetarget protein was visualized with 3,3′-Diaminobenzidine (DAB) as the substrate.Enzymeactivity was assayed by a modified method of McCullough et al.
(McCullough etal., 1971).The reaction mixture containing 100 mM Tris-HCl at pH 7.3, 0.2 mM ZnSO4and 50 µM MTX was equilibrated at 37 ?C for 10 min.
Decrease in the absorbance at 320 nm wasmeasured using Spectrophotometer (T80 UV/VISSpectrometer, PG Instruments). CPG2 activity was assessed using anextinction coefficient of 8300 L mol-1 cm-1 for MTX. Theactivity was calculated in units per milliliter (U/mL).
One Unit is defined asthe amount of the enzyme required to catalyze the hydrolysis of 1µmol MTX per minute at 37 ?C. 2.8.
Quantification of intracellularROS production, GSH content and catalase (CAT) activity The production of intracellular ROS was measured usingDCFH–DA (Jia et al., 2006). DCFH–DA passively enters the cell, whereit reacts with ROS to form a highly fluorescent compound, dichlorofluorescein(DCF). HepG2 cells were seeded in a 6-well plate at 3 ×105 cells for24 h. Cells were treated with CPG2 and TAT-CPG2for 2 h. Cells with or without TAT-CPG2 pre-treatment wereexposed to 10 µM MTX, and ROS detection was evaluatedat 12, 24 and 48 h after treatment.
Following exposure, cells were trypsinizedand washed with the ice-cold PBS. Then, cells were co-incubated with serum-freeDMEM containing10 µM DCFH-DA for 30 min at 37 ?C in the dark. Subsequently, cells were harvested andrinsed with the PBS buffer. Fluorescent intensities were measured using aspectrofluorometer (FluoStar Omega, BMG labtech) at 485 nm excitation/530 nmemission at the end of exposure times. For determining GSH content and CAT activity, HepG2 cellline 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 for12, 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/vglycerol, 1% triton X-100, 1 mM PMSF and 10 µg/ml leupeptin at pH 7.4) and centrifugedat 10,000×g for 15 min. The supernatant was used for determination of the GSHlevel and CAT activity.GSH content was determined using the Ellman method (Ellman, 1959). For assay, 0.
1 ml of the supernatant was added to 0.9ml of 5%trichloroacetic acid (TCA), and centrifuged at 2300 g for 15 min at roomtemperature. 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 GSHlevel was expressed as ?g GSH/mg protein. CAT activity was determined based onthe decomposition of H2O2 (Aebi, 1984). Decrease in the absorbanceat 240 nm was monitored for 2 min using spectrophotometer. Enzyme activity wascalculated using the extinction coefficient of H2O2 (43.6mM?1 cm?1) and expressed as units/mg protein. 2.9. Statistical analysis