Retroviral supernatants were generated by transient transfections of 293T cells with these retroviral constructs and the pCLEco packaging plasmid (Imgenex). Lineage negative BM cells were retrovirally transduced as described [49] and intravenously injected into irradiated (9 Gy) syngeneic hosts. The resultant chimeric mice were analyzed after 8–10 weeks. BW5147a-b- thymoma cells or CD4+ T cells from DO11.10-TCR transgenic mice were transduced using standard spin infection and success- fully transduced cells were identified based on GFP expression. Statistics Statistical analysis was performed using GraphPad Prism software using the two-tailed unpaired Student’s t-test. P- values,0.05 were considered as significant (*), ,0.01 (**), ,0.001 (***). If not otherwise described error bars are calculated from SEM of n = 3 experiments. Supporting Information Figure S1 Mitogenic stimulation of in vitro Th17 polarized cells does not change the expression level of miR-133b or miR-206. Related to Figure 1D and 1E. The same protocol as in Figure 1 was used for the polarization of T cells to the Th17 lineage. 50% of the cells were additionally stimulated with PMA/ionomycin for the last 3 h before harvesting. Expression levels for miR-133b and miR-206 were compared by qRT-PCR relative to Th0 (control). One representative experiment is shown out of two independent experiments with similar results with 2 mice per experiment. Error bars represent SD values. (TIFF) Figure S2 The activation status of CD4+ T cells and cd T cells does not influence miR-133b and miR-206 expres- sion. Related to Figure 2 and Figure 3. (A) CD4+ cells were sorted into naive CD4+ T cells (CD44lo, CD62Lhi) and activated/ memory CD4+ T cells (CD44hi CD62Llo) from lymph node and spleen cells of C57BL/6N mice and compared for their expression levels of miR-133b and miR-206 as described. Shown is one experiment with 5 mice. (B) and (C) cd T cells were sorted into CCR6+, CCR62 CD44hi and CCR62 CD44lo cells from lymph node and spleen cells of TcrdH2BeGFP mice and compared for their expression levels of miR-133b and miR-206. Shown is one representative experiment of two independent experiments with References 1. Behm-Ansmant I, Rehwinkel J, Izaurralde E (2006) MicroRNAs silence gene expression by repressing protein expression and/or by promoting mRNA decay. Cold Spring Harb Symp Quant Biol 71: 523–530. 2. Baltimore D, Boldin MP, O’Connell RM, Rao DS, Taganov KD (2008) MicroRNAs: new regulators of immune cell development and function. Nat Immunol 9: 839–845. 3. Lu LF, Liston A (2009) MicroRNA in the immune system, microRNA as an immune system. Immunology 127: 291–298. 4. Li QJ, Chau J, Ebert PJ, Sylvester G, Min H, et al. (2007) miR-181a is an intrinsic modulator of T cell sensitivity and selection. Cell 129: 147–161. 5. Liu SP, Fu RH, Yu HH, Li KW, Tsai CH, et al. (2009) MicroRNAs regulation modulated self-renewal and lineage differentiation of stem cells. Cell Transplant 18: 1039–1045. 6. Monticelli S, Ansel KM, Xiao C, Socci ND, Krichevsky AM, et al. (2005) MicroRNA profiling of the murine hematopoietic system. Genome Biol 6: R71. 7. Xu S, Cao X (2010) Interleukin-17 and its expanding biological functions. Cell Mol Immunol 7: 164–174. 8. Awasthi A, Kuchroo VK (2009) Th17 cells: from precursors to players in inflammation and infection. Int Immunol 21: 489–498. 9. Wei B, Pei G (2010) microRNAs: critical regulators in Th17 cells and players in diseases. Cell Mol Immunol 7: 175–181. 10. Banerjee A, Schambach F, DeJong CS, Hammond SM, Reiner SL (2010) Micro-RNA-155 inhibits IFN-gamma signaling in CD4+ T cells. Eur J Immunol 40: 225–231. Co-Regulation of miR-133b and miR-206 in the Il17a/f Locus similar results with 4–6 mice per experiment. Error bars show SD values of 3 replicates. (TIFF) Figure S3 Expression of Il17a mRNA in skeletal muscle compared to peripheral lymph node cells and Th17 polarized cells. Skeletal muscle (tibia), peripheral lymph node (inguinal) and Th17 polarized cells were compared for their Il17a mRNA expression by Taqman Real-Time PCR. Shown are the results of 2 independent experiments from 1 mouse per group. (TIFF) Figure S4 No regulation of the predicted target gene Ets1 by miR-133b or miR-206. Luciferase assay using the psiCHECK-2 vector (Promega) into which the Ets1 39-UTR was cloned downstream of Renilla luciferase. By nucleofection with the Amaxa-nucleofection reagent the psiCHECK-2-Ets1 vector was introduced into the BW5147 a-b- cell line that was stably transduced with either the empty MDH1-PGK-GFP2.0 (Addgene) plasmid, the latter plasmid with miR-133b or with miR-206. (TIFF) Figure S5 Proof of principle for the miRNA Real-Time detection system. The miRNAs mmu-miR-133b and mmu- miR-206 were cloned from the BAC AC159614 with the primers mentioned in Materials and Methods. Both miRNAs were introduced into the retroviral vector MDH1-PGK-GFP_2.0 (Addgene) with restriction enzymes EcoRI and XhoI. The respective constructs were then transfected into 3T3 cells via Calcium- Phosphate Transfection Kit (Sigma) and the supernatant was used for the stable transfection of the BW5147a-b- thymoma cell line. (TIFF) Acknowledgments We would like to acknowledge the assistance of the Cell Sorting Core Facility of the Hannover Medical School. We are grateful to Mathias Herberg for excellent animal care. Author Contributions Conceived and designed the experiments: JDH IP AK TK LRW RF. Performed the experiments: JDH KN FP NS VC SS AK IP. Analyzed the data: JDH KN FP NS VC SS AK IP. Contributed reagents/materials/ analysis tools: KN LRW FP TK. Wrote the paper: JDH AK IP. 11. Lu LF, Thai TH, Calado DP, Chaudhry A, Kubo M, et al. (2009) Foxp3- dependent microRNA155 confers competitive fitness to regulatory T cells by targeting SOCS1 protein. Immunity 30: 80–91. 12. Du C, Liu C, Kang J, Zhao G, Ye Z, et al. (2009) MicroRNA miR-326 regulates TH-17 differentiation and is associated with the pathogenesis of multiple sclerosis. Nat Immunol 10: 1252–1259. 13. Ley K, Smith E, Stark MA (2006) IL-17A-producing neutrophil-regulatory Tn lymphocytes. Immunol Res 34: 229–242. 14. Haas JD, Gonzalez FH, Schmitz S, Chennupati V, Fohse L, et al. (2009) CCR6 and NK1.1 distinguish between IL-17A and IFN-gamma-producing gamma- delta effector T cells. Eur J Immunol 39: 3488–3497. 15. Martin B, Hirota K, Cua DJ, Stockinger B, Veldhoen M (2009) Interleukin-17- producing gammadelta T cells selectively expand in response to pathogen products and environmental signals. Immunity 31: 321–330. 16. Michalak P (2008) Coexpression, coregulation, and cofunctionality of neighbor- ing genes in eukaryotic genomes. Genomics 91: 243–248. 17. Purmann A, Toedling J, Schueler M, Carninci P, Lehrach H, et al. (2007) Genomic organization of transcriptomes in mammals: Coregulation and cofunctionality. Genomics 89: 580–587. 18. Williams AH, Valdez G, Moresi V, Qi X, McAnally J, et al. (2009) MicroRNA- 206 delays ALS progression and promotes regeneration of neuromuscular synapses in mice. Science 326: 1549–1554. 19. Gambardella S, Rinaldi F, Lepore SM, Viola A, Loro E, et al. (2010) Overexpression of microRNA-206 in the skeletal muscle from myotonic dystrophy type 1 patients. J Transl Med 8: 48. PLoS ONE | www.plosone.org 10 May 2011 | Volume 6 | Issue 5 | e20171