1. Volume 6, Issue 5, Pages 673-684 (May 2004)
Deacetylase Inhibitors Increase Muscle Cell Size by Promoting Myoblast Recruitment and Fusion through Induction of Follistatin 
Simona Iezzi, Monica Di Padova, Carlo Serra, Giuseppina Caretti, Cristiano Simone, Eric Maklan, Giulia Minetti, Po Zhao, Eric P Hoffman, Pier Lorenzo Puri, Vittorio Sartorelli 
Developmental Cell 
Volume 6, Issue 5, Pages (May 2004)
DOI: /S (04)

2. Figure 1 TSA Increases Myoblasts Recruitment and Fusion
(A) C2C12 myoblasts were exposed to TSA (50 nM) in growth medium and then prompted to differentiate for 48 hr in the absence of TSA. Immunofluorescence and immunoblot were performed with anti-MHC and anti-tubulin monoclonal antibodies. Nuclei were revealed by DAPI staining.
(B) 2-day-old C2C12 myotubes were treated with ara-C (10 μM) for 24 hr and subsequently exposed to either recombinant IGF-1 (10 ng/ml) or TSA (50 nM) for 24 hr and immunostained with anti-MHC and DAPI.
(C) C2C12 myoblasts (1.2 × 105 cells/plate) were either mock-treated or exposed to TSA (50 nM) for 18 hr after which the nuclei, revealed by DAPI staining, were counted (growth medium, GM). For the remaining time points, cells were cultured for an additional 24 hr in GM and then switched to differentiation medium (DM). Nuclei were counted after 24 and 48 hr, respectively. Cells for every experimental point (both control and TSA-treated) were plated and the nuclei counted in duplicate samples. The reported values were obtained after counting at least 5 microscopic fields.
(D) C2C12 myoblasts were cultured as described in (A) and immunostained with anti-MHC. The nuclei were revealed by DAPI staining. The number of nuclei present in MHC+ cells/number of total nuclei per field was determined and expressed as percentage. Nuclei present in 10 microscopic fields were counted.
(E) Human primary skeletal myoblasts were exposed to TSA for 24 hr in GM and then cocultured in DM, in the absence of TSA, with preformed mouse primary myotubes. For TSA-treated cells, two different microscopic fields are shown (TSA). Control represents human primary skeletal myoblasts cells not exposed to TSA and cocultured with preformed mouse primary myotubes. Immunofluorescence was performed with anti-MHC, and nuclei were revealed by DAPI staining. Nuclei of mouse origin (m) show punctuated reactivity to DAPI, while human nuclei (h) display homogenous DAPI staining. Arrows indicate human nuclei in murine myotubes.
(F) Mouse C2C12 myoblasts were exposed to TSA or VPA for 24 hr in growth medium and then cocultured in differentiation medium, in the absence of either TSA or VPA, with preformed human skeletal myotubes. The data are representative of the percentage of the fusion rate of mouse cells into human myotubes (1–2 mouse nuclei per human myotube), as evaluated by counting 6 microscopic fields in duplicate experiments.
Developmental Cell 2004 6, DOI: ( /S (04) )

3. Figure 2 Expression Profiling Analysis of Transcripts Modulated by TSA
(A and B) Microarray analysis of transcripts corresponding to GATA-2 and IGFBP5 in control and TSA-treated cells at different time points (see below).
(C) Myoblasts cultured in wells coated with 0.1% gelatin were exposed to either LY (10 μM), TSA (50 nM), or both LY and TSA for 18 hr in GM and allowed to differentiate in DM for 48 hr before immunostaining with anti-MHC and DAPI.
(D) IL-4 mRNA expression was analyzed by RT-PCR in T lymphocytes activated with CD3/28 and IL-2, unstimulated T lymphocytes, untreated and TSA-treated C2C12 cells.
(E) Experimental design and time frame. Myoblasts were exposed to TSA (50 nM) for 18 hr in GM. The medium was then replaced with DM without TSA and cells harvested at 0, 4, 12, 24, and 48 hr and processed for RNA extraction. Control cells were without TSA.
(F) RT-PCR of transcripts for MHC, pRb, glycogen synthase, and GAPDH in untreated and TSA-treated cells.
(G) Microarray and RT-PCR time course of follistatin and GAPDH transcripts following TSA treatment.
(H) Myoblasts were exposed to either valproic acid (10 mM) or sodium butyrate (10 mM) and allowed to differentiate for 48 hr. Follistatin and GAPDH expression were evaluated by RT-PCR.
Developmental Cell 2004 6, DOI: ( /S (04) )

4. Figure 3
HDAC Inhibitors Activate Follistatin Expression in a Cell Type-Specific Fashion
(A) Primary mouse keratinocytes and C2C12 cells were cultured in either the absence or presence of TSA, sodium butyrate, or VPA for 18 hr and induced to differentiate (D) for 48 hr in the absence of HDAC inhibitors. Untreated, undifferentiated keratinocytes (U) were also included in the experiment. Follistatin and GAPDH expression was evaluated by RT-PCR.
(B) Quantitation of the data presented in (A). The intensity of the signals corresponding to the follistatin and GAPDH bands were determined using the NIH Image software package. The follistatin signals were corrected for the GAPDH signals.
(C) Mouse primary satellite cells were cultured without or with HDAC inhibitors for 18 hr and induced to differentiate (D) for 48 hr in the absence of HDAC inhibitors. U, undifferentiated satellite cells. RT-PCR analysis of follistatin and GAPDH.
(D) Quantitation of the data reported in (C).
(E) C3H10T1/2, NIH 3T3 mouse fibroblasts, 3T3-L1 adipogenic, and MC3T3-E1 osteogenic cells were cultured in either the absence or presence of TSA for 18 hr and for an additional 48 hr in the absence of TSA. RT-PCR analysis of follistatin and GAPDH.
(F) Quantitation of the data reported in (E).
(G) TtT/GF pituitary cells and C2C12 were exposed to either recombinant IL-1β (2 nM) or TSA (50 nM) for 18 hr, cultured for an additional 24 hr, and processed for RT-PCR.
(H) Quantitation of the data reported in (G).
Developmental Cell 2004 6, DOI: ( /S (04) )

5. Figure 4 Follistatin Increases Myoblasts Fusion
(A) Myc-follistatin protein of the expected molecular weight is expressed in myc-follistatin-transduced cells.
(B) Skeletal myoblasts transduced with either a control or myc-follistatin-expressing retrovirus were cultured in DM for 48 hr before immunostaining with anti-MHC.
(C and D) Skeletal myotubes (C) and myoblasts (D) were cultured in the presence of recombinant follistatin (rFS, 0.2 μg/ml) and immunostained for MHC.
(E) The number of nuclei present in MHC+ cells over the total number of nuclei present in at least 10 microscopic filed (expressed as percentage) was determined in control, myc-follistatin-expressing cells, and in myotubes (MT) and myoblasts (MB) cultured in the presence of recombinant follistatin with DAPI staining.
(F) IL-4 mRNA expression was analyzed by RT-PCR in T lymphocytes activated with CD3/28 and IL-2, C2C12 and C2C12 cells expressing myc-follistatin.
Developmental Cell 2004 6, DOI: ( /S (04) )

6. Figure 5
The Follistatin Promoter Is Activated by TSA through MyoD, CREB, and NFAT
(A) The follistatin promoter linked to luciferase (FS-luc) was transfected in C2C12 myoblasts and the cells exposed to TSA or left untreated. The parental luciferase vector (-luc) was employed as control. The cells were differentiated for 48 hr in DM without TSA and harvested, and the luciferase activity was determined.
(B) Schematic representation of the follistatin promoter indicating the presence of binding sites for NFAT, MyoD, and CREB.
(C) The FS-luc reporter and expression vectors for MyoD, CREBY134F, Id1, and A-CREB were transfected into C3H10T1/2 fibroblasts in different combinations. Cells were induced to differentiate for 48 hr and the luciferase activity determined.
(D) The FS-luc reporter and expression vectors for MyoD, NFAT2C, VIVIT, and CREBY134F were transfected into C3H10T1/2 fribroblasts in different combinations.
(E) The FS-luc reporter and expression vectors for either Id1, A-CREB, or VIVIT were transfected into C2C12 myoblasts. The cells were then either exposed to TSA or left untreated and induced to differentiate for 48 hr in the absence of TSA. Every experiment described in this figure was done in triplicate and repeated 3–5 times.
(F) ChIP using control IgG, anti-phospho CREB (phos), or anti-total CREB (total) antibodies. I, input DNA. The follistatin and GAPDH promoter regions were PCR-amplified.
(G) ChIP using either control IgG or anti-NFAT1/2C (N) antibodies. I, input DNA.
(H) Chromatin derived from untreated or TSA-treated C2C12 cells was immunoprecipitated using control IgG, anti-acetylated MyoD (acetyl), or anti-total MyoD (total) antibodies. I, input DNA.
(I) ChIP using control IgG, anti-diacetylated histone H3 (H3), or anti-tetracetylated histone H4 (H4) antibodies. I, input DNA.
Developmental Cell 2004 6, DOI: ( /S (04) )

7. Figure 6
A-CREB, a Dominant-Negative Form of CREB, and VIVIT, a Peptide Blocking the NFAT Activity, Reduce Follistatin Transcription and Render Myoblasts Refractory to TSA
(A) C2C12 myoblasts were infected with adenoviruses coding for either GFP and A-CREB, GFP and CREB Y134F, or GFP alone. After 24 hr, cells were exposed to TSA for an additional 24 hr in GM and then incubated in DM for 12 hr. Extracted RNA was employed in RT-PCR analysis with primers for follistatin or GAPDH.
(B) C2C12 myoblasts were infected with adenoviruses coding for either GFP alone or GFP and A-CREB. After 24 hr, cells were exposed to TSA for an additional 24 hr in GM and then incubated in DM for 36 hr. Infected cells were detected by GFP fluorescence.
(C and D) Retroviruses coding for either GFP alone or GFP and VIVIT were employed to transduce C2C12 myoblasts. After the infection, the cells were either exposed to TSA or left untreated for 18 hr and induced to differentiate in the absence of TSA for an additional 48 hr. Total RNA was isolated and RT-PCR performed with primers for follistatin and GAPDH. In parallel experiments, cells were fixed and visualized by GFP. Nuclei were stained with DAPI.
(E) The number of nuclei present in MHC+ cells over the total number of nuclei present in at least 10 microscopic filed (expressed as percentage) was determined in cells transduced with either GFP or GFP-VIVIT and exposed to TSA.
(F) Anti-MHC immunoblot of total extracts derived from cells transduced with either GFP or VIVIT-GFP retrovirus and exposed or not to TSA.
Developmental Cell 2004 6, DOI: ( /S (04) )

8. Figure 7
Blockade or Knockdown of Follistatin Abrogates the Effects of TSA on Myoblast Fusion
(A) C2C12 myoblasts were cultured in the presence of either TSA, recombinant follistatin, or myostatin. TSA-mediated increased myoblast fusion (TSA) was blocked by recombinant myostatin (100 ng/ml) (myostatin+TSA). Addition of recombinant follistatin (0.2 μg/ml) restored the ability of TSA to augment myoblast fusion in the presence of myostatin (myostatin+rFS+TSA).
(B) Myoblasts were exposed to TSA in the presence of either control IgG (4 μg/ml) or anti-follistatin antibodies (4 μg/ml).
(C) The number of nuclei present in MHC+ cells/number of total nuclei per field was determined and expressed as percentage.
(D) C2C12 myoblasts transduced with either pSUPER.retro (control) or pSUPER.retro bearing an oligonucleotide insert to target follistatin transcripts (FS-RNAi) were exposed to TSA or left untreated before inducing differentiation for 48 hr. Total RNA was isolated and RT-PCR performed with primers for follistatin and GAPDH.
(E) Control and FS-RNAi cells were either exposed to TSA or left untreated and induced to differentiate for 48 hr before immunostaining with anti-MHC.
(F) Anti-MHC and anti-tubulin immunoblot of extracts derived from control and FS RNAi cells exposed to TSA or left unexposed.
(G) The number of nuclei present in MHC+ cells over the total number of nuclei (expressed as percentage) was determined in cells transduced with either control or FS-RNAi retrovirus and exposed to TSA.
Developmental Cell 2004 6, DOI: ( /S (04) )

9. Figure 8
Systemic TSA Delivery in Injured Animals Increases Expression of Follistatin and of Markers of Muscle Regeneration
(A) Three days after injury, RNA was extracted and RT-PCR analysis conducted on crushed tibialis anterior muscles for follistatin, MHC embryonic (emb), MHC perinatal/neonatal (peri), and GAPDH transcripts of untreated (control) or TSA-treated (TSA) animals.
(B) Graphic showing the average of ratios of follistatin, MHCemb, and MHC peri obtained from transcripts derived from either muscle-injured control (C) or muscle-injured and TSA-treated (TSA) animals corrected for their respective GAPDH. Mice (n = 3) for each experimental point were treated for 7 days with either vehicle or TSA.
Developmental Cell 2004 6, DOI: ( /S (04) )