Co-Regulation of miR-133b and miR-206 in the Il17a/f Locus Figure 3. Correlation of IL-17A production and miR-206/133b expression in cd T cells. (A) cd T cells from Tcrd-H2BeGFP mice were sorted into a CCR6+ and CCR62 populations and analyzed for expression of miR-133b and miR-206 by qRT-PCR. Dot plots show post-sort analysis of one representative experiment. (B) cd T cells were sorted into GFP+ (Rorct+) and GFP2 (Rorct2) populations from heterozygous Rorct reporter mice and analyzed as in (A). (C) cd T cells from Tcrd-H2BeGFP mice were sorted into CD272 and CD27+ populations and analyzed as in (A). Dot plots show post-sort analysis of one representative experiment. Values are plotted as fold increase ( = ratio) compared to the respective negative population. The graphs show representative experiments from n = 3 independent experiments with similar results. Error bars represent SD values of triplicates from one experiment with 4–6 mice. doi:10.1371/journal.pone.0020171.g003 Figure 4. The Th17 polarizing cytokine IL-23 promotes expression of miR-206/133b as well as secretion of IL-17A. (A, B) Spleen and peripheral lymph node cells were isolated from DO11.10 mice, cocultered with sex-matched BALB/c irradiated feeder cells and polarized to either Th1 or treated with TGF-b, IL-6, IL-23, IL-1b, IL-21 and TNF-a in various combinations (4cytokines = TGF-b, IL-6, IL-23 and IL- 1b). Values show fold increase ( = ratio) compared to cells cultured only with Ova323–329/antibodies. Expression levels of miR-133b (A) and miR- 206 (B) were analyzed by qRT-PCR. (C) Secreted IL-17A was determined in culture supernatants of each condition from the cells in (A) and (B) by ELISA. Values show absolute amounts of IL-17A in the cell culture supernatant in ng/ml. Error bars show 6SEM of n = 3 experiments with 2–3 mice per experiment. (D), (E) Scatter plot of IL-17A protein concentration versus relative expression of miR-133b (D) and miR-206 (E) and correlation coefficient for ELISA compared to qRT-PCR. doi:10.1371/journal.pone.0020171.g004 PLoS ONE | www.plosone.org 5 May 2011 | Volume 6 | Issue 5 | e20171