PMC:3824683 / 28127-39183 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3824683","sourcedb":"PMC","sourceid":"3824683","source_url":"http://www.ncbi.nlm.nih.gov/pmc/3824683","text":"Materials and Methods\n\nCell culture and reagents\nAll the experiments were performed either in SVGA astrocytes, a clone of SVG astrocytes, or primary HFAs. HFAs were obtained from aborted fetuses and \u003e98% of the cells were found to be positive for GFAP (data not shown). Both the SVGA and the primary HFA cells were maintained at 37 °C in a humidified chamber with 5% CO2. The cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1% L-glutamine, 1% sodium bicarbonate, 1% non-essential amino acids, and 0.1% gentamicin. All cells were cultured in 12-well plates and allowed to adhere overnight before any treatment was performed.\nMA was purchased from Sigma-Aldrich (St. Louis, MO, USA). The plasmid expressing gp120 (pSyngp120 JR-FL) was originally constructed by Drs. Park and Seed (Department of Molecular Biology, and Melvin and Barbara Nessel Gene Therapy Center, Massachusetts General Hospital, Boston, MA, USA). Recombinant protein gp120IIIB and pSyngp120 JR-FL plasmids were obtained from the NIH AIDS Research and Reference Program (Germantown, MD, USA). DAS was obtained from Fisher Scientific (Pittsburgh, PA, USA). DPI, DFO, OP, TROLOX, vit. E, fluoxetine, and paroxetine were purchased from Sigma-Aldrich. The treatments with antagonist were performed 1 h before the stimulus (MA and/or gp120) and maintained throughout the length of exposure. Specific antibodies against ferritin heavy chain (B12), CYP2E1 (H-21), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (FL-335) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), antibody against caspase-3, which detects procaspase3 (35 kDa) and cleaved caspase-3 (17 kDa) was purchased from Cell Signaling Technology (Danvers, MA, USA) and antibody against CYP2D6 and CYP2B6 were purchased from Abcam (Cambridge, MA, USA). Specific siRNA for CYP2E1, CYP2B6, CYP2D6, NOX2, NOX4, and control siRNAs were purchased from Dhramacon (Thermo Fisher Scientific Inc., Waltham, MA, USA).\n\nTransfection\nSVGA astrocytes were transfected with 2 μg plasmid encoding gp120 (pSyngp120 JR-FL) using Lipofectamine 2000 (Invitrogen Inc., Carlsbad, CA, USA) as discussed previously.5, 6, 14, 41 Briefly, 3 × 105 SVGA cells per well were seeded in a 12-well plate and incubated overnight to allow them to adhere. The following day, the complete medium was removed and the cells were washed two times with PBS, followed by incubation with serum-free medium. The transfection reagents were added to the cells that were transfected and incubated for 5 h. After 5 h, the cells were washed two times with PBS and incubated with the complete medium for a total duration of 24 h. Cells transfected with empty vector were used as controls in all the transfection experiments. For the treatments with inhibitor, SVGA astrocytes were pretreated with the antagonist 1 h before the transfection in serum-free medium and the antagonist was maintained throughout the duration of transfection.\nIn experiments involving siRNA transfections, SVGA cells were seeded at 8 × 105 cells per well in a six-well plate. The following day, cells were washed two times with PBS and transfected with 20 pmolof specific siRNA against NOX4, CYBB (NOX2), CYP2E1, CYP2B6, CYP2D6, or control siRNA for 48 h. The cells were then recounted and 2.75 × 105 cells per well were seeded in a 12-well plate. These gene-depleted cells were then exposed to MA and/or gp120 for 24 h and ROS was measured. The efficiency of gene knockdown was confirmed using western blotting.\n\nReal-time reverse transcriptase-polymerase chain reaction\nTo measure the mRNA expression levels of various CYPs, the cells were either treated with 500 μM MA and/or transfected with plasmid encoding gp120. Upon termination of the treatment, total RNA was isolated using Qiagen RNeasy Mini Kit (Qiagen, Valencia, CA, USA). The RNA (120 ng) was reverse transcribed into cDNA at 37 °C for 60 min using a 2-step TaqMan Gene Expression Kit (Applied Biosystems, Foster City, CA, USA). The cDNA obtained was amplified for GAPDH (4333764F), CYP1A1 (Hs01054794_m1), CYP2B6 (Hs03044636_m1), CYP2E1 (Hs00559367_m1), CYP3A4 (HS00430021_m1), CYP2D6 (Hs00164385_m1), and CYP2A6 (Hs0071162_m1) using the probe mix obtained from Applied Biosystems as per the manufacturer's protocol. The expression values for various CYPs were normalized using GAPDH as a housekeeping gene. Relative fold expressions for various genes were analyzed using the 2−ΔΔCt method.\n\nMeasurement of ROS production\nTo measure the oxidative stress produced in the astrocytes, the cells were treated with various agents for appropriate duration. After the termination of treatments, the cells were washed two times with PBS and incubated with 5 μM 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) (Molecular Probes, Carlsbad, CA, USA) for 30 min in serum-free medium at 37 °C. The cells were then washed two times with PBS to remove unloaded dye and the images were taken using Leica fluorescent microscope (DMI 3000B; Leica Microsystems Inc., Buffalo Grove, IL, USA). Unstained cells were used as experimental controls and the fluorescent images were obtained using excitation and emission wavelengths at 485 and 535 nm, respectively.\nTo obtain the fluorescence intensity corresponding to ROS, the cells were collected and acquired using FITC wavelengths on FACSCanto II flow cytometer (BD Biosciences, San Jose, CA, USA). The ROS production was measured using unstained cells as negative controls and cells treated with 500 μM H2O2 as positive controls. The mean fluorescence intensities were compared between different treatments.\n\nWestern blotting\nThe protein levels were measured by analyses of western blots using whole-cell lysates obtained from astrocytes treated with appropriate reagents. Briefly, the cells were lysed using radioimmunoprecipitation assay (RIPA) buffer (Boston BioProducts, Ashland, MA, USA) supplemented with HaltTM Protease Inhibitor Cocktail (Pierce Biotechnology, Rockford, IL, USA). The protein concentrations in the lysates were measured using BCA Protein Assay Kit (Pierce Biotechnology). The protein samples (30 μg) were electrophoresed on a 10% polyacrylamide gel and transferred to PVDF membrane. Finally, the membrane was probed with appropriate primary and secondary antibodies for caspase-3, ferritin heavy chain (Ferritin HC), NOX2, NOX4, CYP2B6, CYP2E1, and CYP2D6 to measure their expression levels. The bands were detected using BM Chemiluminescence Western Blotting Substrate (POD) (Roche Applied Sciences, Indianapolis, IN, USA). The bands were analyzed using FlourChem HD2 software (Alpha Innotech, San Leandro, CA, USA) and the intensities were normalized using GAPDH as loading control.\n\nCell survival assay (MTT assay)\nThe cell viability was measured using a colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Briefly, 2 × 105 astrocyte cells were seeded in 12-well plates and treated with either the inhibitor or saline with subsequent treatment with MA and/or gp120 for 48 h. Upon termination of the treatments, the medium was replaced with 0.2 mM MTT containing medium and incubated at 37 °C for 4 h. The medium was carefully removed and purple formazan crystals were dissolved in 500 μl DMSO containing 125 μl of Sorenson's glycine buffer. The color formation was measured using Benchmark Microplate Reader (Bio-Rad Laboratories, Hercules, CA, USA) with absorbance at 570 nm and reference at 650 nm.\n\nTUNEL assay\nThe measurement of cell viability was also correlated with DNA damage using TUNEL assay as per the manufacturer's protocol (GenScript, Piscataway, NJ, USA). Briefly, 2 × 105 cells per well were seeded in 12-well plates and the cells were exposed to the treatments with appropriate compounds. Upon treatments, the cells were air-dried for 5 min and fixed (4% paraformaldehyde in PBS) for 30 min at room temperature. After fixation, they were incubated overnight at 4 °C and permeabilized using 1% Triton X-100. The cells were then blocked in 3% H2O2 in methanol and labeled with TUNEL reaction mixture (biotin-11-dUTP and TdT) and streptavidin-FITC. The smear of cells were washed with PBS to remove excess of labeling reagents and incubated with POD-conjugated anti-FITC substrate. Finally, 3,3′-diaminobenzidine (DAB) working reagent was added and the cells were observed under the light microscope. The images were taken using Leica DMI 300B (Leica Microsystems Inc., Buffalo Grove, IL, USA).\n\nCaspase-3 activity assay\nThe caspase-3 activity was measured using caspase-3 colorimetric assay kit (BioVision Inc., Milpitas, CA, USA) as per the manufacturer's protocol. Briefly, apoptosis was induced as per the treatments under investigation and the cells were lysed using lysis buffer. The protein concentration in the lysates was measured using BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL, USA) and 50 μl of lysates containing 200 μg protein was used for caspase-3 activity. The lysates were mixed with 2 × reaction buffer containing 10 μM DTT and DEVD-pNA substrate and incubated at 37 °C for 2 h. Finally, the absorbance was measured using Benchmark Microplate Reader (Bio-Rad Laboratories) at 405 nm and the caspase activity was calculated with relative standard curve obtained using substrate alone.\n\nProtein carbonylation assay\nThe measurement of carbonylated protein was performed using protein carbonyl colorimetric assay kit (Cayman Chemicals, San Francisco, CA, USA) as per the manufacturer's protocol. Briefly, the cells were treated and lysed with RIPA lysis buffer. The protein samples were checked for the absence of nucleic acid contamination and 200 μg proteins were used for the assay. The lysates were mixed with DNPH (2,4-dinitrophenylhydrazine) and incubated in the dark for 1 h followed by consecutive treatments with 20% and 10% trichloroacetic acid (TCA). This was followed by three washes with ethanol/ethyl acetate (1 : 1 mixture and finally the pellet was resuspended in guanidine hydrochloride. The absorbance of the supernatants was measured at 370 nm using a microplate reader and carbonylated protein content was calculated.\n\nStatistical analysis\nThe statistical analysis was performed to represent the data in mean±S.E. values. Results were based on at least three separate experiments unless specified with each experiment performed in triplicates. For the comparison between mock/control group, treatments were performed using two-tailed Student's t-test to calculate P-values, and P-value ≤0.05 was considered statistically significant. The P-value ≤0.05 was indicated by ‘*' and ≤0.01 was indicated by ‘**' in the bar graphs. Experiments involving two variables were analyzed using two-way ANOVA to address whether they interact with each other in a synergistic manner. To examine dose-dependent effect of the inhibitors, results were analyzed using P-value for trend test using general linear model. No adjustments were made in the statistical analysis for multiple comparisons. 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