Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS) and penicillin/ streptomysin were obtained from Gibco BRL (Gland Island, NY, USA). Cadmium chloride (Cd), NAC (N-acetyl-L-cysteine) were from Sigma (St. Louis, MO, USA). BAPTA-AM [1,2-bis(2-aminophenoxy) ethane-N,N,N’,N’-tetraacetic acid tetrakis (acetoxy methyl ester)] were obtained from Calbiochem-Novabiochem (San Diego, CA, USA). GADD153 antibodies were obtained from abcam (Cambridge, CB4 OFL, UK). Rb and Bax antibodies were obtained from Santa Cruz Biotechnology (Santa cruz, CA, USA). Luciferase assay kit was purchased from Promega (Madison, WI, USA). 2′,7′-dichlorodihydrofluorescein diacetate (2′,7′-dichlorofluorescein diacetate, H2DCFDA) were obtained from Molecular Probes (Eugene, OR, USA). pGL2 Bak promoter construct was kindly provided by Dr. Yong-Sung Juhnn(Seoul National University, Korea), pcDNA3-GADD153 expression construct was kindly provided by Dr. Dae-Ghon Kim(Chonbuk National University, Korea).
Human neuroblastoma (SH-SY5Y) cells (ATCC, Rockville, MD, USA) were cultured in DMEM supplemented with 10% FBS, 100 Units/mL penicillin, and 100 μg/mL streptomycin. Cells were maintained in a 37°C /5% CO2 incubator.
Measurement of reactive oxygen species
SH-SY5Y cells were treated with BAPTA-AM for 30 min and Cd treated for 11 hr 30 min. Cells were washed with phosphate-buffered saline (PBS), harvested using trypsin and washed with PBS. Resuspend cells in H2DCFDA (final concentration 20 μM) at 37°C incubator for 30 min. Cells were washed with PBS and then resuspend in PBS. ROS analyzed by flow cytometry (Beckman culter, Brea, CA, USA).
Measurement of apoptosis by propidium iodide staining
SH-SY5Y cells were treated with Cd for 12 hour that they washed with PBS, harvested using trypsin. Cells were stained with propidium iodide (PI; 5 μg/mL) in PBS. They were analyzed by flow cytometry, and the extent of apoptosis was determined based on the sub-G1 population.
Transfection and luciferase assay
SH-SY5Y cells were transfected with plasmid using Lipofectamine 2000 Reagent (Invitrogen, Camarillo, CA, USA) according to the manufacturer’s recommendation. Cells were incubated for 24 hr in 37°C 5% CO2 incubator. Luciferase activities were assayed using luciferase assay kit (Promega, Madison, WI, USA), according to the manufacturer’s instructions.
Western blot analysis
Cells were lysed in RIPA buffer [50 mM Tris–HCl, pH 7.5, 1% NP-40, 0.5% sodium deoxycholic acid, 0.1% SDS, and protease inhibitors cocktail (sigma, St. Louis, MO, USA)] on ice for 20 min. Lysates were centrifuged at 12,000 × g for 15 min. Protein (60 μg) was separated by 10% SDS-PAGE, and the proteins were transferred onto a 0.45 μm PVDF membrane (Milipore, Bedford, MA, USA). After the membrane was blocked with 5% fat-free milk in TBST buffer (25 mM Tris–HCl, pH 7.4, 137 mM NaCl, 5 mM KCl, and 0.1% Tween 20) for 3 hr, it was incubated with the primary antibodies at 4°C overnight and secondary antibodies for 2 hr. All antibodies were used at a dilution of 1:1000. Membrane were developed using an enhanced SuperSignal West Femto Chemiluminescent Substrate (Thermo, Waltham, MA, USA).
Cells were treated with Cd for 12 hour and then washed two times with PBS, harvested using cell scraper. Cells were centrifuged at 300 × g for 4 min, resuspended buffer A (10 Mm HEPES, pH 7.5, 10 Mm KCl, 1 mM DTT, 1 mM PMSF, protease inhibitors cocktail) and incubated for 15 min on ice. Supernatant (cytosol) was collected by centrifuged at 4°C, 1500 × g for 5 min after 10 min incubation on ice with 10% NP-40. Pellet was washed two times with buffer A and then resuspended buffer B (buffer A + 0.4 M NaCl) for 30 min on ice. Supernatant (nuclear) was collected by centrifuged at 4°C, 15000 × g for 10 min.
DAPI staining & annexin V assay
Cells were treated with an NAC for 1 h and then incubated with Cd for 12 h. After treatment with Cd, cells were washed with PBS, fixed for 30 min with 4% paraformaldehyde (PFA) prepared in PBS, followed by incubation with 4′,6-diamindine-2-phenilindole (DAPI; 10 μg/mL) for 30 min. They were analyzed by flow cytometry. Apoptotic cells with condensed or fragmented nuclei were visualized under a fluorescence microscope (Olympus Optical Co., Melville, NY, USA). Apoptotic cells were detected by annexin V assay kit (Molecular Probes, Inc., Eugene, OR, USA), according to the manufacturer’s instructions.
SH-SY5Y cells were routinely cultured and treated with 25 μM Cd for 12 h. Cells were fixed by adding 37% formaldehyde (final concentration 1%) directly to culture medium and incubate for 10 min. Cells were sonicated condition for 15 sec for18 time, 2 min-intervals, setting 35% output with micro-tip to keep sample on ice. Cells were detected by Chromatin immunoprecipitation assay kit (Millipore, Temecula, CA, USA), according to the manufacturer’s instructions. ChIP products using specific antibodies directed against pre-immune IgG (Santa cruze, Califonia, DA, USA) or GADD153. The purified DNA products were amplified target protein-DNA specific primer (Sense primer: 5′-AATCTAGTATTAGTATTCCCCA-3′, Anti-sense primer: 3′-ACCATTCTGGCTAACATGGTGA-5′) and non-specific primer (Sense primer: 5′-GTCTGCAT CCGGTGGCCACA-3′, Anti-sense primer: AACCCGGT CCTAGGGCCGTC).
Data are expressed as the mean ± SD of experiments performed in triplicate and replicated at least three times. Data were evaluated with one-way analysis of variance (ANOVA) followed by Student’s t-test. Statistically significant differences are reported as *p < 0.05 or **p < 0.01. Data with values of p < 0.05 were generally accepted as statistically significant.