Abstract:

Dataset includes 1 excel file that contains the relative fold difference of HepG2 cells exposed to different chemicals for 24 hours. The starting concentration was 2 × 10^6^ cells per 60 cm^2^ tissue culture dish. After incubation, cells were lysed. Protein extracts from cell lysis were separated with SDS gel electrophoresis and Peroxidase activity was detected with western blot analysis using a ChemiDoc XRS+System (BioRad) #1708265. This dataset supports the publication: Alqassim, A. Y., Wilson, M. J., Wickliffe, J. K., Pangeni, D., Overton, E. B., & Miller, C. A. (2019). Aryl hydrocarbon receptor signaling, toxicity, and gene expression responses to mono‐methylchrysenes. Environmental Toxicology, 34(9), 992–1000. doi:10.1002/tox.22770

Suggested Citation:

Ahmad Alqassim, Charles Miller. 2019. Protein expression induced by chrysene and methylchrysenes in HepG2 cells. Distributed by: GRIIDC, Harte Research Institute, Texas A&M University–Corpus Christi. doi:10.7266/n7-t4q0-0t85

Publications:

Alqassim, A. Y., Wilson, M. J., Wickliffe, J. K., Pangeni, D., Overton, E. B., & Miller, C. A. (2019). Aryl hydrocarbon receptor signaling, toxicity, and gene expression responses to mono‐methylchrysenes. Environmental Toxicology, 34(9), 992–1000. doi:10.1002/tox.22770

Purpose:

To detect the protein expression for HepG2 cells post chemical exposure.

Data Parameters and Units:

This dataset contains one excel file with three spreadsheets titled after the Antigen used in the experiment - CYP1A1, CYP1B1, and CYP1A2. 0 = chrysene 1 = 1-methylchrysene 2 = 2-methylchrysene 3 = 3-methylchrysene 4 = 4-methylchrysene 5 = 5-methylchrysene 5 = 5-methylchrysene 6 = 6-methylchrysene 7 = DMSO The peroxidase activity from the antibodies was detected using ChemiDoc XRS+System (BioRad) #1708265 and evaluated using NIH ImageJ software.

Methods:

HepG2 cells were incubated in a Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, 50 μg/mL streptomycin, 50 U/mL penicillin and propagated in an incubator with 5% CO2 at 37°C. HepG2 cells were then treated with 10 μM chrysene, or its methylated derivatives according to the toxic and non-toxic concentrations identified in the SRB toxicity test. Cells were seeded at 2 × 106 cells per 60 cm^2^ tissue culture dish. The cells were treated 24 h later and exposures lasted for 24 h. After treatment, the medium was removed and the cells were washed with ice-cold PBS and emptied. 250 µl of Laemmli sample buffer, consisting of 2.1% SDS, 20% glycerol, 65.8 mM tris, 0.01% bromophenol blue, and 5% mercaptoethanol at a pH ~6.8 (1:1), was placed on the plate. The viscous lysates were collected in microcentrifuge tubes and placed on ice. The samples were transferred to boiling water for 60 s and then pulsed for 10 seconds by high-frequency sound waves (20 to 30 kHz) with an ultrasonic microtip probe to reduce the viscosity. The proteins were loaded onto a 4-15% gradient SDS-polyacrylamide gel. Approximately 15 μL lysate was loaded per well. Colored BioRad protein molecular-weight (MW) markers (10 μL/lane) were added to determine protein mobility. Electrophoresis was conducted using a running buffer consisting of 25 mM tris, 192 mM glycine, 0.1% sodium dodecyl sulfate (SDS), at pH~8.3. The gel was run with 50 V for 5 minutes and for 150 V until the dye front ran off the bottom of the gel. The gel was removed and its proteins transferred to a nitrocellulose membrane using a buffer that consisted of 25 mM tris, 192 mM glycine, 20% methanol, at pH~8.3. The protein transfer took 60 minutes at 100 V. The nitrocellulose membranes were blocked with bovine serum albumin (BSA) or nonfat dry milk 5% (vol/vol) in tris buffered saline (TBS, pH= 7) for 1 hour and then washed three times with TBS 10 minutes each. The antibodies used (CYP1A1, CYP1B1, CYP1A2, and GAPDH) were manufactured by ThermoFisher Scientific and diluted by 1:200; 1:200; 1:100 and 1:1000 respectively. After incubation for 24 h, cells were washed with TBS as above. The same process was repeated with the secondary peroxidase-conjugated antibody (Mahmood and Yang, 2012; Towbin et al., 1979; and Bradford, 1976). The membrane was rinsed with three washes in TBS. Pierce ECL Plus Western Blotting Substrate was prepared by mixing substrate A and substrate B in a 40:1 ratio. The substrate was added to the membrane and incubated for 5 min in plastic wrap under dim light. Peroxidase activity from the secondary antibody was detected using ChemiDoc XRS+System (BioRad) #1708265 and evaluated using NIH ImageJ software.

Instruments:

Peroxidase activity from the secondary antibody was detected using ChemiDoc XRS+System (BioRad) #1708265 and evaluated using NIH ImageJ software.

Provenance and Historical References:

Mahmood T, Yang P-C. (2012). Western Blot: Technique, Theory, and Trouble Shooting. North American Journal of Medical Sciences, 4(9), 429- 434. doi: 10.4103/1947-2714.100998. Towbin H, Staehelin T, Gordon J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A, 76(9), 4350-4. doi: 10.1073/pnas.76.9.4350. Bradford MM. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem, 72, 248-54. doi: 10.1016/0003-2697(76)90527-3.

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