Cell lines, antibodies and plasmids
H1299 non-small cell lung cancer cells, p53-intact and p53-deficient HCT116 colon cancer cells, DU145 prostate cancer cells and p53
-null mouse embryonic fibroblasts (MEFs) were maintained in DMEM medium with 10% FBS. Antibodies against HA (Covance, Berkeley, CA), Flag (Sigma, St Louis, MO), SUMO-1 (Zymed Laboratories, San Francisco, CA), SIRT1 (Santa Cruz Biotechnology, Santa Cruz, CA), p53 (DO-1; BD Biosciences, San Jose, CA), acetyl-Lys 382 p53 (Trevigen, Gaithersburg, MD), and SENP1 (Abgent, San Diego, CA) were all obtained from commercial sources. Except where indicated, all expression vectors encode human proteins. Expression vectors for HA–SIRT1 (ref. 25
), Flag–SIRT1 (ref. 25
), mouse Flag–SIRT1 (ref. 14
, Flag–SENP1 (refs 28, 36
, His–SUMO-1 and HA-SUMO-1 (ref. 31
have been described previously. SIRT1ΔCT
was generated by inserting the BamH
1 fragment of human SIRT1
cDNA into the pcDNA3–HA vector. For SIRT1 expression in Escherichia coli, SIRT1
cDNA was inserted into the BamH
1 sites of the pET-30a vector. SIRT1 mutants were generated by site-directed mutagenesis using the Quick Change kit from Stratagene (La Jolla, CA). The sequences of the mutagenesis primers were: 5′ -TAATGAAGCTATATCTGTGAGACAGGAAGTAAC- 3′ for Lys 734; 5′-TGG TTCTAGTACTGGGGAGAGAAATGAAAGAACT- 3′ for Lys 610; and 5′-GTT GTT AAT GAA GCT ATA GCT GTA AAA CAG GAA TTG ACA G- 3′ for mouse SIRT1VK
Whole-cell extracts were prepared by sonication of cells in a buffer containing 20 mM Tris–HCl at pH 7.5, 0.5% NP-40, 150 mM NaCl, 3 mM EDTA, 3 mM EGTA, 10 μg ml−1 aprotinin, 10 μg ml−1 leupeptin, 10 mM benzamidine and 1 mM phenylmethylsulfonyl fluoride. The extracts were incubated with 4 μg antibody for 4 h at 4 °C and subsequently with protein G agarose beads for an additional 4 h. After washing five times with lysis buffer, the immunoprecipitates were heated in SDS–PAGE sample buffer. Proteins in the supernatant were resolved on 8% SDS–PAGE gels and transferred to nitrocellulose membranes. The membranes were probed with antibodies as indicated and proteins recognized by the antibodies were visualized by enhanced chemiluminesence. To detect p53 acetylation at Lys 382, approximately 1 × 106 H1299 cells were plated on 100-mm dishes. Twenty-four hours later, the cells were transfected with 4 μg HA–p53 and additional plasmids as indicated. Eighteen hours post-transfection, cells were treated with 0.5 mM H2O2 or exposed to 0.3 J UV radiation (UV crosslinker, Stratagene) if necessary. Cells were harvested 6 h later, and amounts of acetylated p53 were determined by immunoprecipitation or immunoblotting of cell extracts with antibody to acetyl-Lys 382 p53.
To detect SIRT1 sumoylation, H1299 or DU145 cells were transfected with 4 μg Sumo-1 and SIRT1 plasmids 24 h after plating on 100-mm dishes. If needed, cells were then treated with 0.5 mM H2O2 or exposed to 0.3 J UV radiation 18 h post transfection and harvested 6 h later for immunoprecipation and immunoblotting analysis.
For immunofluorescence analyses, H1299 cells transfected with wild-type SIRT1 and SIRT1K734R on coverslips were washed three times with PBS and fixed in 2% paraformaldehyde for 15 min at room temperature. After additional washing with PBS, fixed cells were made permeable with a solution containing 1% Triton X-100 and 1% BSA. Permeabilized cells were incubated with mouse anti-SIRT1 or rabbit anti-SENP1 antibodies for 2 h at room temperature followed by incubation with goat anti-mouse IgG conjugated with Alexa Fluor 594 (red) or anti-rabbit IgG conjugated with fluorescein isothiocyanate (FITC, green; Molecular Probes, Carlsbad, CA) for 1 h at room temperature. Cells were then washed three times in PBS and stained with DAPI in anti-fade mounting medium. Fluorescent microscopic images were obtained with a Leica confocal laser scanning microscope or a Leitz Orthoplan 2 microscope.
In vitro sumoylation, deacetylase and desumoylation assays
To measure SIRT1 sumoylation in vitro, His–SIRT1 was produced using the E. coli T7 S30 Extract System (Promega, Madison, WI) and purified with the μMACX His-tagged protein isolation kit (Miltenyi Biotec Inc, Auburn, CA) following the manufacturer's protocols. In brief, after five high-stringency washes with buffer containing 50 mM Tris at pH 8.0, 500 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate and 0.1% SDS, and rinsing in 20 mM Tris at pH 8.0, His–SIRT1 was eluted from the beads with the buffer for the native condition and dialysed against sumoylation buffer containing 20 mM HEPES at pH 7.5, 5 mM MgCl2, 2 mM ATP. Purified SIRT1 protein (1 μg) was added to a 20 μl reaction in an in vitro sumoylation system (LAE Biotech International, Rockville, MD) containing different sumoylation components as indicated. Reactions were carried out at 37 °C for 2 h, or as indicated. Reaction mixtures were boiled in SDS sample buffer and separated by SDS-PAGE and SIRT1 protein was detected by immunoblotting with an anti-SIRT1 antibody.
For deacetylase assays, the SIRT1 sumoylation reactions were dialysed against deacetylase assay buffer containing 50 mM Tris at pH. 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2 and 1 mg ml−1 BSA and deacetylase activity was determined using the SIRT1 fluorimetric drug discovery kit (AK-555; BIOMOL International, Plymouth Meeting, PA) following the manufacturer's protocol. In brief, sumoylated SIRT1 was incubated in the assay buffer with the acetylated p53 peptide Arg-His-His-Lys (ε-acetyl) and NAD+ at 37 °C. After 30 min, the deacetylase reactions were stopped by adding Developer II. The deacetylation of the substrate sensitizes it to Developer II which then generates a fluorophore. The fluorophore was then excited with 360 nm light and the emission light at 460 nm detected on a multi-detection microplate reader (Synergy HT; Bio-Tek Instruments, Winoosky, VT).
To assay the deacetylase activity of SIRT1 isolated from cells, HA-tagged human SIRT1 or Flag-tagged mouse SIRT1 was transfected into H1299 cells and purified on an anti-HA or M2 antibody-conjugated affinity column. Cell lysates were loaded onto an anti-HA or M2 affinity column equilibrated with a buffer containing 20 mM Tris–HCl at pH 7.5, 100 mM NaCl, 0.1 mM EDTA, and the columns were washed three times with buffer containing 20 mM Tris–Hcl at pH 7.5 and 1 M NaCl. Proteins were eluted in equilibration buffer containing 1 mg ml−1 HA or 100 μg ml−1 Flag peptide. The deacetylase activity of the eluted SIRT1 proteins was assayed using the fluorimetric drug discovery kit.
For in vitro
desumoylation, HA–SIRT1 conjugated to His–Sumo-1 was purified from HEK293 cells expressing HA–SIRT1 and His–Sumo-1 using the μMACX His-tagged protein isolation kit. After five high stringency washes of the beads, the sumoylated SIRT1 protein was eluted from the beads with the buffer for native conditions and dialysed against desumoylation buffer containing 10 mM Tris–HCl at pH 8.0, 150 mM NaCl and 1 mM DTT. Wild-type and inactive mutant Flag–SENP1 proteins were produced in an in vitro
TNT translation system (Promega) and purified using M2 affinity beads. After five high stringency washes, Flag–SENP1 was eluted from the beads with a solution containing 100 μg ml−1
Flag peptide. Eluted proteins were dialysed against desumoylation buffer. SENP1 desumoylation assays were performed as previously described37
Transfections and reporter assays
p53-null MEFs were plated in 12-well plates in DMEM containing 10% FBS at 1 × 105 cells per well. One day after plating, cells were transfected by Lipofectamine Plus following the protocol provided by Gibco–BRL. Cell lysates were prepared by directly adding lysis buffer (25 mM Tris-phosphate at pH 7.8, 2 mM DTT, 2 mM 1,2-diaminocyclohexane-N′,N′,N′,N′-tetraacetic acid, 10% glycerol and 0.2% Triton X-100) to the cells on ice. Luciferase activity was determined using the dual luciferase assay system from Promega. The reporter activity was normalized to the activity of renilla luciferase to minimize the artifacts caused by variation transfection efficiency.
Ni-bead pulldown assays
To detect SIRT1 conjugates with His–SUMO-1, transfected cells were divided into two aliquots. One aliquot was immunoblotted to determine the level of protein expression. The second aliquot was lysed under denaturing conditions in a buffer containing 10 mM Tris–HCl at pH 8.0, 6 M guanidinium–HCl, 100 mM Na2HPO4−NaH2PO4, 5 mM imidazole and 10 mM β-mercaptoethanol, and incubated with Ni2+-NTA beads (Qiagen, Valencia, CA) for 4 h at room temperature. The beads were washed with lysis buffer followed by two sequential washes with a buffer containing 10 mM Tris–HCl at pH 8.0, 8 M urea, 100 mM Na2PO4/−NaH2PO4 and 10 mM β-mercaptoethanol, and a buffer containing 10 mM Tris–HCl at pH 6.3, 8 M urea, 100 mM Na2PO4−NaH2PO4 and 10 mM β-mercaptoethanol. His–Sumo-1 conjugated SIRT1 was eluted from the beads with a buffer containing 150 mM Tris–HCl at pH 6.7, 200 mM imidazole, 30% glycerol, 0.72 M β-mercaptoethanol and 5% SDS. The eluted proteins were immunoblotted with anti-Flag or HA antibodies.
To measure p53-induced caspase-3 activation, cells were permeabilized, fixed and stained for active caspase-3 with FITC-conjugated anti-caspase-3 antibody. The percentage of apoptotic cells was determined by flow cytometry according to the instructions of the active caspase-3 apoptosis kit I (BD Biosciences).
To measure cell death, cells (1 × 106 per 100-mm dish) were stained with propidium iodide, and DNA content was determined by flow cytometry on a Becton-Dickinson FACScan. As alternative approaches, apoptosis was also analysed by the cell death detection ELISAPLUS (Roche, Indianapolis, IN), according to the manufacturer's protocol. The assay system quantifies the amount of histone-coupled DNA fragments by measuring absorbance at 405 nm.
To measure the effect of SIRT1
siRNA on UV radiation- and H2
-induced cell death, H1299 cells were transfected with SENP1
in pSuppressorNeo vector (IMGENEX Corporation, San Diego, CA) and selected against G418. SIRT1
siRNA was delivered into the cells by retroviral infection followed by selection with puromycin, as previously described29
. SMART pool siRNA specific to p73
and scrambled controls were synthesized by Dharmacon and transfected into cells by lipofectamine 2000 following manufacturer's protocols. After treatment with UV radiation and H2
, cell apoptosis were analysed by cell death detection ELISAPLUS