(A) The WT and mutant 3′-UTR sequences of the human genes CTCF (target to miR-27b), NCOA3 (miR-206), ELK3 (miR-106b), AHR (miR-203), and ELF2 (miR-23b) were cotransfected with miR mimics or a scrambled sequence (con-miR). Relative light units (RLUs) were normalized to the respective control group and are presented as relative luciferase activity. Results of 3 independent experiments with n = 3 per group are shown. (B) Relative expression of target miRs in in vitro–polarized murine Th2 cells was determined by real-time RT-PCR and normalized to naive CD4⁺ T cells. Graph summarizing results of 3 independent experiments with n = 2–3 per group. (C) Relative mRNA expression, depicted as fold change, of the selected miR targets Ctcf, Ncoa3, Elk3, Ahr, and Elf2 in murine Th2 cells normalized to naive CD4⁺ T cells in n = 4–5 samples. (D) Graph summarizing the results for IL-13⁺ cells after nucleofecting miR inhibitors and miR mimics as indicated in the table below the bar graph and antagonizing the IDEAL (Impact of Differential Expression Across Layers) predictions. In each condition, the con-miR transfection contained the equal amount and composition of control sequences (see Methods section). Values were normalized to the respective con-miR samples and summarize 4 independent experiments with n = 2–3 replicates per experiment. (E) Representative histogram showing the effect of antagonizing the miR expression pattern in Th2 cells on IL-13 expression. A miR cocktail containing anti–miR-27b, miR-206 mimic, miR-106b mimic, miR-203 mimic, and anti–miR-23b was nucleofected. IL-13⁺ cells were determined by flow cytometry (blue dashed line). The effect was controlled by nucleofecting the respective amount of scrambled miR mimics/inhibitors (con-miR, red line; see Methods). Shaded histogram indicates samples stained with isotype control antibody. Values summarize 4–5 independent experiments with n = 2–3 replicates per experiment. (F) Twenty-four hours after nucleofection, cells were stimulated with anti-CD3/anti-CD28 for 24 hours and cytokines were measured in culture supernatant. Concentrations were normalized to con-miR sample. Graph summarizes results of 4 independent experiments with n = 2–3 replicates per group and experiment. All graphs with quantitative data show mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 by 2-tailed Mann-Whitney U test (D–G).