A. MATERIALS
TPA, insulin, and EGF were purchased from Sigma (St. Louis, MO, USA), and antibodies against pErk1/2, Erk1/2, PEA-15pS104, PEA-15, pAktS473, 53BP1 and Akt were from Cell Signaling (Danvers, MA, USA). Antibodies to p53, p21WAF1, Lamin B1, HA and α-tubulin were from Santa Cruz (Santa Cruz, CA, USA), and anti-PEA-15pS116 was from Invitrogen. Anti-PML antibody was from Abcam (Cambridge, MA, USA) and anti-histone H3K9 dimethylated (H3K9m2) antibody was purchased from Millipore (Billerica, MA, USA).
B. METHODS
1. Cells culture
HDF cells were isolated in our laboratory from foreskin of a 4 year-old boy (Lim et al., 2000;
Kwak et al., 2004), and the primary culture was maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Invitrogen/GIBCO, Grand Island, NY, USA). Number of population doublings and their doubling times were calculated by the published equations (Kim et al., 2009). HDF young cells used in this study represent doubling time of around 26 h, the mid-old and the old cells indicate doubling times of around 4–10 days and over 14 days, respectively. Cells were incubated in 5% CO2 at 37 °C.
2. Senescence-associated b-gal assay
Cells washed twice in PBS and fixed for 5 min in 4% formaldehyde were incubated at 37 °C with staining solution containing 1.0 mg of 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal) per ml of dimethylformamide, 40 mM citric acid/sodium phosphate (pH 6.0), 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl and 2 mM MgCl2. Staining was evident in 2-4 h and maximum in 12-16 h.
3. Immunoprecipitation (IP) and immunoblot analyses
IP was performed with cell lysates (~1.0 mg protein) by the standard method in modified RIPA buffer without 0.1% SDS. Cell lysates pre-cleared with protein G-agarose beads for 1 h were precipitated with targeted antibodies for 4 h at 4 °C. The immunoprecipitates were washed 5
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times with IP buffer, and then subjected to immunoblot analyses in RIPA buffer. Proteins bands were resolved on 10-15% SDS-PAGE, and transferred to polyvinylidene fluoride (or nitrocellulose) membranes before hybridization with a targeted antibody overnight at 4 °C. The membranes were incubated with horseradish peroxidase-conjugated secondary antibodies for 1 h, and ECL (Amersham, Pharmacia Biotech, Piscataway, NJ, USA) kit was employed to visualize protein bands.
4. Cell fractionation
Cells were harvested with PBS and then lysed in TD buffer [25 mM Tris base (pH 8.0), 2.0 mM MgCl2, 0.25% v/v Nonident P40, 0.5 mM DTT, 1.0 μg/ml leupeptin, 100 μg/ml PMSF, 1.0 mM Na3VO4, 1.0 mM NaF] for 5 min at room temperature (RT). After centrifugation at 12,000 g for 5 min, the supernatant was collected as cytoplasmic fraction, and the pellets were resuspended in BL buffer [10 mM Tris (pH 8.0), 0.4 M LiCl, 0.5 mM DTT, 1.0 μg/ml leupeptin, 100 μg/ml PMSF, 1.0 mM Na3VO4, 1.0 mM NaF] with vigorous vortex, and cell debris was removed by centrifugation for 20 min.
5. Immunocytochemistry
Cells on cover slips were washed with PBS before fixation with 4% paraformaldehyde for 15 min, permeabilized with 0.05% Triton X-100, and then incubated with 3% bovine serum albumin in 0.05% Triton X-100 for 2 h. Primary antibody was applied overnight at 4 °C, secondary antibody at 4 °C for 2 h, and then stained with 4% 6-diamidino-2-phenylindole (2.0 mg/ml) for 10 min at RT before mounting. Expressions of pErk1/2 and PEA-15 were detected using polyclonal primary antibodies along with Alexa 488 or Alexa 555 conjugated goat-anti rabbit IgG as a secondary antibody. Data visualized under fluorescence microscope were photographed by AxioVision image acquisition and analyzed by software package (Carl Zeiss MicroImaging GmbH).
6. BrdU incorporation assay
Cells were treated with TPA for 30 min after transfection with either siControl or siPEA-15 for 48 h, pre-labeled with 10 μM BrdU (Sigma) for 2 h, fixed in 4% paraformaldehyde solution, and incubated in 2 M HCl for 30 min. pH was raised with 0.1 M sodium borate (pH 8.5) for 2 min. Immunocytochemistry steps were followed by using anti-BrdU FITC-labeled antibody
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(eBioscience, San 125 Diego, CA, USA), and positive cells were counted under fluorescence microscope.
7. Cell cycle analysis
Cells were treated with TPA for 30 min after transfection with siPEA-15 for 48 h. They were then trypsinized and fixed before re-suspended in propidium iodide solution. DNA analysis was performed by flow cytometry (BD Biosciences, San Jose, CA, USA).
8. Cell proliferation assay
HDF mid-old cells (1.0 x 105) were plated in 60 mm dish 24 hours before transfection for 4 h with siRNAs (40nM) against PEA-15 or its scrambled sequences (siControl), and then the culture medium was changed with fresh one before incubation for 4 more days. Treatment of HDF cells with either DMSO (0.01%) or TPA (50 ng/ml) before the 4 day incubation was employed as the negative and positive controls of the experiment, respectively. All of the cells were harvested in 2 and 4 days after the treatment, and the numbers of cells were then counted by using Hemocytometer (Marienfeld-Superior, Germany). Changes of cell numbers were evaluated by Student's t-test. To monitor cell proliferation assay, HDF young cells (0.8 x 105) were plated in 60 mm dish, and then their proliferation activity was also monitored every 2 days.
9. Real-time PCR analysis
Total cellular RNAs were extracted with RNAiso Plus (TaKaRa Bio, Ohtsu, Japan), and cDNAs were synthesized with 1.0 μg of RNA and reverse transcription kit (Invitrogen). The cDNAs were used for real time PCR analysis with specific primers and SYBR Green PCR Master Mix (Applied Biosystems) under the conditions: Initial activation at 95 °C for 15 min, followed by 40 cycles at 95 °C for 20s and 60 °C for 40s. Primers used for c-fos amplification were 5’-TGACTGATACACTCCAAGCGGA-3 and 5’-CAGGTCATCAGGGATCTTGCA-3’
and for β-actin were 5’-CCCTGGCACCCAGCAC-3’ and 5’-GCCGATCCACACGGAGTAC-3’
10. siRNA transfection
siRNAs against PEA-15 (siPEA-15) were purchased from Santa Cruz Biotechnology and control siRNAs from DHARMACON (Lafayette, CO, USA). Cells on a cover slip were transfected with siRNAs and Oligofectamine (Invitrogen) for 4-6 h. In 48 h, the cells were
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treated with mitogen for 30 min before subjected to immunocytochemistry or immunoblot analyses.
11. Statistical analysis
All data were presented as means ±S.D and analyzed by one-way ANOVA for comparison between multiple groups or Student’s t-test using SPSS. Probability value less than 0.05 was considered as statistically significant.
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