TopHat 1.1.4 was used to align the sequenced reads back to the mouse reference genome (UCSC mm9). The normalized fragment density was calculated by counting the fragments per kilobase of genomic regions of interests (coding sequences (CDS), introns, 59 UTR, 39 UTR, and non-coding (nc) exons) per million mapped reads. In both NC (A) and miR-29 (B) samples, the majority of the RNA-seq reads fall into the transcript regions (CDs, 59UTR, and 39UTR), demonstrating good specificity for mRNAs. (C) The expression of ECM genes, Col 1a1, Col 1a2, Col 3a1, Vimentin, as well as Lims1 in NC and miR-29 expressing cells as revealed by RNA-seq. (D) miR-29 over-expression in 10T1/2 cells (Fibroblasts) leads to the down- regulation of ECM synthesis. (E) miR-29 over-expression in 293 cells leads to the down-regulation of Col 3a1 expression. (TIF) Figure S2 miR-29 down-regulates Collagens in C2C12 myoblasts. Col 1A1, Col 1A2 or Col 3A1 39UTR reporter plasmid was transfected into C2C12 cells with indicated miRNA oligos. Luciferase activities were determined at 48 h post- transfection and normalized to b-Galactosidase protein. Relative activity is shown with respect to control cells where normalized luciferase values were set to 1. The data represents the average of three independent experiments 6 S.D. (TIF) Figure S3 TGF-b inhibits miR-29 during myogenic and fibrogenic differentiation of C2C12 cells. (A) C2C12 cells were treated with 5 ng/ml of TGF-b in differentiation medium (DM) for 0, 3 and 6 days. RNAs were isolated for qRT-PCR measurement of the expressions of Myogenin, MyHC, a-Actin, and Troponin normalized to GAPDH. Expression folds are shown with respect to 0 hr cells where normalized copy numbers were set to 1. Data are plotted as mean 6 S.D. (B) Cell morphology was visualized under phase contrast. Bars = 50 mm. (C) C2C12 cells were transfected with 0.2 mg of Troponin-Luc reporter plasmids along with Renilla reporter plasmid and treated with TGF-b for 48 hrs at which time luciferase activities were determined and normalized to Renilla luciferase activity. Relative light unit (RLU) is shown with respect to untreated cells where normalized luciferase values were set to 1. The data represent the average of three independent experiments 6 S.D. (D) C2C12 cells were treated with TGF-b for 3 days. Proteins were isolated for Western measurement of the expression of Cadherin-11, FSP1 (Fibroblast- specific protein-1), Transgelin using Tubulin as a loading control. (E) C2C12 cells were treated with TGF-b in DM for 0, 3, 6 days. Total RNAs were isolated for qRT-PCR measurement of the expression of Col 1A1, Col 1A2, Col 3A1, a-SMA or VIM normalized to GAPDH. Expression folds are shown with respect to 0 hr cells where normalized copy numbers were set to 1. Data are plotted as mean 6 S.D. (F). C2C12 cells were treated with TGF-b for 0, 1, 2 or 3 days and the viable cell number was counted. Proliferation folds are shown with respect to day 0 cells where normalized proliferation fold were set to 1. Data are plotted References 1. Gregory RI, Chendrimada TP, Cooch N, Shiekhattar R (2005) Human RISC couples microRNA biogenesis and posttranscriptional gene silencing. Cell 123: 631–640. 2. Hutvagner G (2005) Small RNA asymmetry in RNAi: function in RISC assembly and gene regulation. FEBS Lett 579: 5850–5857. 3. Sabourin LA, Rudnicki MA (2000) The molecular regulation of myogenesis. Clin Genet 57: 16–25. 4. Wang H, Sun H, Guttridge DC (2009) microRNAs: novel components in a muscle gene regulatory network. Cell Cycle 8: 1833–1837. 5. Wang H, Garzon R, Sun H, Ladner KJ, Singh R, et al. (2008) NF-kappaB-YY1- miR-29 regulatory circuitry in skeletal myogenesis and rhabdomyosarcoma. Cancer Cell 14: 369–381. miR-29 in Myoblast Conversion to Myofibroblast as mean 6 S.D. (G) Left: C2C12 cells treated with TGF-b were seeded on 6-well plate with 100% confluent monolayer and a ‘‘wound’’ was induced. Phase-contrast pictures of the wound were taken at 0, 3, 6 and 9 hr. Right: The percentage of wound closure was quantified at each indicated time point. (H) C2C12 cells were treated with or without TGF-b in DM for four days. Cells were fixed and stained for a-SMA (green). DAPI (blue) staining was also performed to visualize the nuclei. Photos were taken by confocal scanning microscope. (I) NC or miR-29 stable cells were untreated (2) or treated (+) with TGF-b in DM for the indicated time intervals at which time RNAs were isolated for qRT-PCR analysis of Myogenin, Troponin, a-Actin and MyHC. Expression folds are shown with respect to NC cells without TGF-b treatment where normalized copy numbers were set to 1. Data are plotted as mean 6 S.D. (TIF) Figure S4 miR-29 inhibits Smad3 expression. Basal and phosphorylated (p) Smad3 levels were examined in C2C12 cells over-expressing miR-29 or with miR-29 knock-down by Anti-miR oligos. Tubulin was used as a loading control. The quantification of Smad3 or p-Smad3/Tubulin was performed using ImageJ 1.43u (National Institutes of Health, USA). The expression folds are shown with respect to control where normalized expression fold were set to 1. (TIF) Table S1 List of up-regulated genes in miR-29 express- ing C2C12 cells. (PDF) Table S2 List of down-regulated genes in expressing C2C12 cells. (PDF) miR-29 Table S3 Functional annotation clustering of up-regu- lated genes in miR-29 expressing C2C12 cells. (XLS) Table S4 Functional annotation clustering of down- regulated genes in miR-29 expressing C2C12 cells. (XLS) Acknowledgments We thank Prof. Dennis Y. M. Lo and Prof. Rossa W. K. Chiu for granting us the access to GAIIx sequencer and their generous support throughout the course of this study. We thank Prof. Huiyao Lan for providing Smad3 and Smad7 knockout mice as well as his expert assistance. We are also grateful to Prof. Zhenguo Wu for critical review of our manuscript. Author Contributions Conceived and designed the experiments: HW. Performed the experi- ments: LZ LL LW HW. Analyzed the data: PJ HS. Wrote the paper: HW LZ. 6. Katagiri T, Yamaguchi A, Komaki M, Abe E, Takahashi N, et al. (1994) Bone morphogenetic protein-2 converts the differentiation pathway of C2C12 myoblasts into the osteoblast lineage. J Cell Biol 127: 1755–1766. 7. Hu E, Tontonoz P, Spiegelman BM (1995) Transdifferentiation of myoblasts by the adipogenic transcription factors PPAR gamma and C/EBP alpha. Proc Natl Acad Sci U S A 92: 9856–9860. 8. Li Y, Foster W, Deasy BM, Chan Y, Prisk V, et al. (2004) Transforming growth factor-beta1 induces the differentiation of myogenic cells into fibrotic cells in injured skeletal muscle: a key event in muscle fibrogenesis. Am J Pathol 164: 1007–1019. 9. Li Y, Huard J (2002) Differentiation of muscle-derived cells into myofibroblasts in injured skeletal muscle. Am J Pathol 161: 895–907. PLoS ONE | www.plosone.org 13 March 2012 | Volume 7 | Issue 3 | e33766