Fig. 2. SIRT1 interacts with PML and is recruited to the PML NBs. (A) Endogenous SIRT1 co-immunoprecipitates with PML IV. HeLa cells were transfected with either pcDNA3Gal4PML IV or empty pcDNA3Gal4, and the cell lysates were immunoprecipitated (IP) with anti-Gal4 antibody. The complexes were resolved by SDS–PAGE and analyzed by western blotting with anti-SIRT1 and anti-Gal4 antibodies as indicated. Endogenous SIRT1 and Gal4PML IV are indicated by arrows. (B) Endogenous SIRT1 and PML interact. HeLa cells were treated with 1 µM As₂O₃ for 4 h and whole-cell extracts (WCE) were immunoprecipitated with anti-PML or an irrelevant antibody. The complexes were resolved by SDS–PAGE and analyzed by western blotting with anti-SIRT1 antibody. Endogenous SIRT1 is indicated by an arrow. (C) Overexpression of PML IV in MEFs or WI38 cells results in the accumulation of both endogenous and GFP–SIRT1 in the NBs. MEFs were infected with PINCO-SIRT1 (panels 1–3) or PINCO-SIRT1 and pBABE-PML IV (panels 4–6). PML was detected with an anti-PML antibody followed by incubation with a Cy3-conjugated secondary antibody (red), while GFP–SIRT1 was revealed by the intrinsic green fluorescence of GFP. Merging of two colours results in yellow signal corresponding to co- localized proteins. MEFs were transfected with pcDNA3GFPPML IV (panels 7–9). WI38 cells were infected with pBABE-PML IV (panels 10–12) or pBABE (panels 13–15). Endogenous SIRT1 or mSir2α staining was revealed with anti-SIRT1 antibody followed by incubation with a Cy3- conjugated secondary antibody (red), while PML staining was monitored either by GFP fluorescence or with an anti-PML antibody followed by incubation with an Alexa 488-conjugated secondary antibody (green). (D) Expression of oncogenic Ras in MEFs recruits mSir2α to the NBs. MEFs were infected with pBABE-RasV12 (panels 1–3) or pBABE (panels 4–6). Endogenous PML and mSir2α stainings were performed as in (C).

Fig. 4. SIRT1 interacts with p53 in vitro and in vivo. (A) Pull-down experiment performed with in vitro translated (IVT) SIRT1 and GST–p53 (lane 3) or different GST–p53 fusions expressing different portions of the protein as indicated (lanes 4–6). Complexes were resolved by SDS–PAGE and visualized by autoradiography. IVT SIRT1 is indicated by an arrow. The exposure time of the input lane is half that of the binding reactions. (B and C) Whole-cell extracts (WCE) from untreated 293T cells or HCT116 UV-irradiated cells (20J/m² for 5 or 20 h) were immunoprecipitated (IP) with either anti-SIRT1 or pre-immune sera, and the immunocomplexes analyzed by western blotting with anti-p53 (DO-1) antibody. Different exposures are shown in (C) in order to visualize p53 immunoprecipitated in the various samples. The asterisks indicate the Ig heavy chain.

Fig. 6. SIRT1 represses p53-mediated transactivation. (A) A luciferase reporter assay was performed in a fibroblast cell line derived from MEFs. Cells were transfected with 2 µg of PGluc (lanes 1–6) and 2.5 µg of pHKp53 (lanes 2–4), 1.5 µg of pcDNA3.1SIRT1 (lanes 3 and 5) or 1.5 µg of pcDNA3.1SIRT1H363Y (lanes 4 and 6). Relative luciferase units shown are the mean value of triplicates. (B) 293T cells were transfected with 0.1 µg of mdm2-luc (lanes 1–6) and 0.025 µg of pHKp53 (lanes 2–4), 0.5 µg of pcDNA3.1SIRT1 (lanes 3 and 5) or 0.5 µg of pcDNA3.1SIRT1H363Y (lanes 4 and 6). Relative luciferase units shown are the mean value of triplicates.

Fig. 8. Proposed model for the rescue of PML-induced premature senescence by SIRT1 overexpression. Upregulation of PML promotes acetylation of p53 by CBP and enhances its activity, leading to the expression of p53 target genes and the onset of senescence. We propose that SIRT1 expression antagonizes PML-induced acetylation of p53, thus modulating its activity and exerting a negative action on the induction of senescence.

Fig. 1. SIRT1 is an active nuclear NAD-dependent HDAC. (A) Equivalent amounts of wild-type His-SIRT1 or mutant His-SIRT1H363Y were tested for HDAC activity in the presence (full column) or absence (open column) of 1 mM NAD⁺. Histone deacetylase activity is given as radioactivity (c.p.m.) of [³H]acetate released from an acetylated histone H4 peptide. Control assays containing only acetylated H4 peptide without any recombinant protein were performed. (B) NAD-dependent HDAC assay performed essentially as above in which GST–SIRT1 was tested for activity in the absence (2) or presence of either 2 µM TSA (3) or 5 mM nicotinamide (4). All reactions contain identical amounts of GST–SIRT1. (C) Asynchronous HeLa cells were analyzed for endogenous SIRT1 expression using an anti-SIRT1 antibody followed by incubation with an Alexa 488-conjugated secondary antibody (green). Nuclear DNA was stained with propidium iodide (red). Note the high level of propidium iodide staining in the nucleoli.

Fig. 3. PML, SIRT1 and p53 co-localize in the NBs. U20S cells were either transfected with pcDNA3-PML IV (panels 1–7) or un-treated (panels 9–15). PML staining with mouse monoclonal PGM3 is shown in blue (AMCA), endogenous p53 staining with goat polyclonal anti-p53 in red (Cy3) and endogenous SIRT1 staining with rabbit anti-sirt1 in green (Alexa 488). Co-localization is shown in yellow (p53 and SIRT1), light blue (PML and SIRT1), violet (PML and p53) and white (triple co-localization). Panels 8 and 16 show the normalized intensity profiles along the lines showed in the insert respectively: p53 (red line), SIRT1 (green line) and PML (blue line). Blue peaks identify PML accumulation in the NBs. Peak distribution in panel 8 supports triple co-localization of p53, SIRT1 and PML.

Fig. 5. SIRT1 can deacetylate p53 in vitro and in vivo. (A) Equivalent amounts of GST (lane 1) or GST–p53 (lanes 2–4) were acetylated in vitro by bacterially expressed p300 in the presence of [¹⁴C]acetylCoA. Recombinant SIRT1 was then added to the reactions in the presence (lane 4) or absence of 1 mM NAD⁺ (lane 3). Two micromolar TSA was added to all reactions. The samples were resolved by SDS–PAGE and acetylated p53 was visualized by autoradiography. Ponceau staining shows the total levels of GST–p53 used in the reactions. (B) An in vitro deacetylation reaction with GST–p53 and SIRT1 was performed as in (A), followed by western blot analysis with a specific antibody for acetylated lysine 382 (αAcK382p53). Acetylated GST–p53 is shown with an arrow. (C) 293T cells were co-transfected with combinations of the expression plasmids pcDNA3.1SIRT1, pcDNA3.1SIRT1H363Y and pcDNA3GAL4CBP(1099–1758) as indicated, or with empty vector controls (lane 1). Twenty-four hours post-transfection, whole-cell lysates were immunoprecipitated with DO-1 antibody and the complexes were analyzed by SDS–PAGE. The acetylation status of endogenous p53 was visualized using a specific antibody for acetylated lysine 382 (αAcK382p53). The bottom panel shows the total amount of p53 immunoprecipitated in each sample. (D) 293T cells were transfected with either pcDNA3.1SIRT1 (lane 3) or with empty vector control (lanes 1 and 2). Twenty-four hours post-transfection, cells were UV-irradiated (150 J/m², lanes 2 and 3) and after 5 h, whole-cell extracts were prepared from control and UV-treated cells. Immunoprecipitations and western blotting were performed as in (C). (E) MCF7 cells were UV-irradiated (50 J/m², lanes 2 and 3), and when indicated, nicotinamide was added to the media immediately following irradiation. Whole-cell extracts were prepared from control and UV-treated cells after 20 h. Immunoprecipitations and western blotting were performed as in (C). (F) SIRT1 overexpression in MEFs inhibits p53 acetylation. Immortalized MEFs were transfected with PINCO-SIRT1 and pcDNA3-p53. p53 overexpression was analyzed by DO-1 staining followed by Alexa 350-conjugated secondary antibody (blue, panel 1) while GFP fluorescence revealed GFP–SIRT1 staining (panel 3). Acetylated p53 was analyzed with anti-acetylated p53 antibody followed by Cy3-conjugated secondary antibody (red, panel 2). Mean fluorescence value of p53 and acetyl-p53 signals were measured in cells expressing low (gfpSIRT1–) and medium to high (gfpSIRT1+) GFPSIRT1 levels (panel 4; Student’s t-test, p = 0.002). The graph shows the normalized mean fluoresence intensities.

Fig. 7. SIRT1 antagonizes PML-induced cellular senescence. (A) Overexpression of SIRT1 in primary MEFs rescues PML IV-induced cell growth arrest. Wild-type MEFs were infected with the indicated retroviruses. Infected cells were selected, inoculated at 5 × 10⁴ cells per well and counted for the subsequent 8 days. Day 0 is the first day after selection. The cell number at each time point shown on the growth curves represents the mean value of duplicate wells. (B) SIRT1 rescues a PML-induced senescent phenotype. WI38 cells were infected with the indicated retroviruses. Cells were selected, cultured for 8 days and then examined for GFP positivity, BrdU incorporation and acidic β-galactosidase activity. The BrdU-positive cells and β-galactosidase positive cells are shown as a percentage of GFP-positive cells. (C) SIRT1 inhibits PML-induced p53 acetylation. MEFs were infected with the indicated retroviruses. Following selection, cells were cultured for 4 days, lysates were prepared and p53 was immunoprecipitated with a rabbit polyclonal anti-p53 antibody. The acetylation status of endogenous p53 was visualized using a specific antibody for acetylated lysine 382 (αAcK382p53), while total p53 was revealed with an anti-p53 antibody (Ab-7). p53–/– cells are included as a negative control for the p53 immunoprecipitation.