1. Volume 12, Issue 1, Pages 51-62 (July 2003)
Sir2 Regulates Skeletal Muscle Differentiation as a Potential Sensor of the Redox State 
Marcella Fulco, R.Louis Schiltz, Simona Iezzi, M.Todd King, Po Zhao, Yoshihiro Kashiwaya, Eric Hoffman, Richard L. Veech, Vittorio Sartorelli 
Molecular Cell 
Volume 12, Issue 1, Pages (July 2003)
DOI: /S (03)

2. Figure 1
Sir2 Inhibitors Activate and Sir2 Represses Transcription Mediated by Muscle-Specific Enhancers
(A) C2C12 cells were transfected with the indicated reporters and exposed to increasing concentrations of either sirtinol (25–50 μM) or M-15 (50–100 μM).
(B) C2C12 or 10T1/2 fibroblasts were transfected with the indicated reporter constructs, and expression vector for MyoD (only for 10T1/2 cells since C2C12 contain endogenous MyoD) or MEF2C, and increasing concentrations (0.25–1 μg) of a Sir2 expression vector.
(C) C2C12 cells were transfected with MCK-luc and exposed to NAM (10 mM) before determining luciferase activity. Every experiment was done in triplicate points.
Molecular Cell  , 51-62DOI: ( /S (03) )

3. Figure 2
The Deacetylase Activity of Sir2 Is Necessary to Repress Muscle Gene Expression and Differentiation
(A) C2C12 cells were transduced with either a control or a retrovirus coding for FLAG-Sir2 or for FLAG-Sir2H355Y and cultured for 36 hr in differentiation medium (DM).
(B) Time course of MHC and myogenin expression in cells transduced with either control or Sir2 retroviruses.
(C) Expression of MHC, myogenin, FLAG-Sir2 and tubulin in control, Sir2-, and Sir2H355Y-transduced muscle cells cultured in DM for 36 hr.
(D) Control, Sir2-, and Sir2H355Y-expressing cells were cultured in DM for 36 hr either in the presence or absence of NAM (10 mM) and their extracts analyzed for MHC, FLAG-Sir2, and tubulin expression.
(E) Human primary skeletal myoblasts were transduced with either a control or Sir2-expressing retrovirus and cultured in DM for 36 hr. Immunofluorescence was conducted with antibodies against MHC (green), anti-murine Sir2 (red), and DAPI (blue). Anti-murine Sir2 does not cross-react with human Sir2 (data not shown).
Molecular Cell  , 51-62DOI: ( /S (03) )

4. Figure 3 Interaction of Sir2 and PCAF/GCN5 Acetyltransferases
(A) Myc-tagged Sir2 and FLAG-tagged PCAF were expressed in 293 cells and cell extracts immunoprecipitated with a myc antibody. The immunoprecipitated material was analyzed by immunoblotting for the presence of FLAG-PCAF with the M2 FLAG antibody. WCE, whole-cell extract.
(B) The experiment was conducted as described in (A) except that immunoprecipitation was performed with M2 FLAG antibody and immunoblot analysis with a myc antibody.
(C) Interaction of endogenous PCAF and Sir2. Extracts from undifferentiated (MB) or differentiated (MT, 48 hr in DM) muscle cells were immunoprecipitated with either control IgG or anti-Sir2 and immunoblotted with anti-PCAF and anti-Sir2 antibodies.
(D) In vitro interaction of Sir2 and PCAF. Baculovirus-produced and purified FLAG-PCAF and bacterially expressed and purified His-Sir2 proteins were incubated and immunoprecipitated with either control IgG or anti-histidine antibody. Immunoblot was performed with M2 FLAG and anti-histidine antibodies.
(E) Myc-Sir2 and FLAG-hGCN5 liter or FLAG-hGCN5-S were expressed in 293 cells and cell extracts immunoprecipitated with M2 FLAG antibody. Immunoblot was performed with myc and M2 FLAG antibodies.
(F) Schematic representation of PCAF, hGCN5-L/PCAF-B, and hGCN5-S proteins.
(G) The core domain of Sir2 mediates interaction with PCAF. Several Myc-Sir2 deletion mutants were coexpressed with FLAG-PCAF in 293 cells. Cell extracts were immunoprecipitated with M2 FLAG antibody and immunoblotted with myc and M2 FLAG antibodies (left panel). The right panel shows the input Sir2 proteins. The oval surrounds Sir2 deletion 513–735, which fails to interact with PCAF.
(H) Schematic summary of the regions of Sir2 investigated and their abilities to interact with PCAF.
Molecular Cell  , 51-62DOI: ( /S (03) )

5. Figure 4
Interactions of Sir2, PCAF, and MyoD and Deacetylation of PCAF and MyoD by Sir2
(A) MyoD, PCAF, and Sir2 were expressed in 293 cells and cell extracts immunoprecipitated with a Sir2 antibody. Immunoblotting was performed with anti-PCAF, MyoD, and Sir2 antibodies. WCE, whole-cell extract. Note the presence of endogenous PCAF (lane 2).
(B) The core domain of Sir2 is sufficient to mediate formation of a complex containing PCAF and MyoD. The core domain (aa 236–513) or the C terminus (aa 513–736) of Sir2 fused to a Myc epitope was coexpressed with FLAG-PCAF and FLAG-MyoD and cell extracts immunoprecipitated with a Myc antibody. Immunoblotting was performed with MyoD, PCAF, and Sir2 antibodies. The asterisk (*) indicates the position of IgG chains.
(C) Extracts obtained from muscle cells at different stages of differentiation (subconfluent cells grown in GM [growth medium]; DM [differentiation medium] for 24 and 48 hr) were immunoprecipitated with either control IgG or MyoD antibodies. Immunoblotting was performed with Sir2, PCAF, and MyoD antibodies.
(D) FLAG-MyoD, FLAG-PCAF, and Myc-Sir2 were expressed in 293 cells in either the absence or presence of NAM (10 mM). Cell extracts were immunoprecipitated with M2 FLAG antibody and immunoblotted with anti-acetyl lysine antiserum (to detect acetylated PCAF) and anti-acetylated MyoD affinity-purified antiserum (to detect acetylated MyoD), anti-PCAF (to detect total PCAF), and anti-MyoD antibody (to detect total MyoD).
(E) Baculovirus-produced and purified FLAG-PCAF and bacterially expressed and purified His-MyoD were employed in an in vitro acetylation reaction. Deacetylation was initiated by adding purified recombinant Sir2 either in the absence or presence of NAD+.
Molecular Cell  , 51-62DOI: ( /S (03) )

6. Figure 5
Chromatin Immunoprecipitation of Two Muscle Regulatory Regions with Antibodies against MyoD, PCAF, and Sir2 and Differentiation Profile of Muscle Cells with RNA Interference of Sir2
(A) Chromatin derived from cells transduced with either a Sir2 wt or Sir2H355Y mutant retrovirus and cultured in differentiation medium for 24 hr was immunoprecipitated with control IgG (I), MyoD (M), Sir2 (S), or PCAF (P) antibodies and subjected to PCR with primers either corresponding to the myogenin and MHC promoter regions containing MyoD binding sites or amplifying a MHC coding region (primer sequences are reported in Supplemental Data at IN, input DNA. A DNA titration was performed to demonstrate a linear detection range.
(B) Chromatin obtained as described in (A) was immunoprecipitated with anti-acetyl K9 and K14 histone H3 antibody (Ac H3) and subjected to PCR with the same primers employed in (A).
(C) Cell extracts derived from muscle cells grown in either growth medium (GM) or differentiation medium (DM) for 0–48 hr were analyzed by immunoblotting for the presence of the indicated proteins.
(D) C2C12 cells were transduced with either a control or a retrovirus expressing a short hairpin (sh) RNA targeting Sir2 and prompted to differentiate for 36 hr. Immunofluorescence was performed with a MHC antibody. Immunoblot of Sir2, MHC, and tubulin in control (C) and sh RNA Sir2 (sh Sir2)-expressing cells.
(E) Human primary skeletal muscle cells were transduced with either a control or a retrovirus expressing a short hairpin (sh) RNA targeting Sir2 and prompted to differentiate for 48 hr. Immunofluorescence was performed with a MHC antibody. Immunoblot of Sir2, MHC, and tubulin in control (C) and sh RNA Sir2-expressing cells.
Molecular Cell  , 51-62DOI: ( /S (03) )

7. Figure 6
The [NAD+]/[NADH] Ratio Influences Muscle Gene Expression through Sir2
(A) Estimation of the free cytosolic [NAD+]/[NADH] ratio was determined by measurement of the [lactate] and [pyruvate] in extracts derived from cells cultured in differentiation medium for 0, 12, 24, and 48 hr. The numbers in parentheses are the standard deviations.
(B) Cells were cultured either in the absence or presence of increasing concentrations of L-lactate (2.5, 5, and 10 mM) or pyruvate (10, 25, and 30 mM) for 48 hr and MHC and tubulin expression evaluated by immunoblot.
(C) Measurement of the [NAD+]/[NADH] ratio obtained from the extracts employed for the experiment described in (B).
(D) Cells transduced with either control or sh RNA Sir2 retrovirus were cultured in either the absence or presence of increasing concentrations of pyruvate (15 and 20 mM) for 48 hr and MHC, Sir2, and tubulin expression determined by Western blot. Fold reduction refers to the reduction of the signals corresponding to MHC upon pyruvate treatment as determined by scanning and measuring the signal intensity with the NIH Image 1.6 software package.
(E) Control and C2C12 sh RNA Sir2 cells were cultured in either the absence or presence of pyruvate (20 mM) and immunostained with a MHC antibody.
(F) C2C12 cells were cultured with increasing concentrations of pyruvate (15, 20, 25 mM) in the absence (control) or presence of NAM (10 mM) and their extracts analyzed for MHC and tubulin content by immunoblot.
Molecular Cell  , 51-62DOI: ( /S (03) )