Figure 2.

Alloantigen-induced microRNAs (miRNAs) target transforming growth factor-beta (TGF-β) family members and signaling molecules. Total RNA was extracted from purified CD4⁺ cells in the draining lymph node (dLN) of syn- or allografted mice 10 days post transplant. (A) Validation of microarray results through quantitative real-time (qRT)-PCR. (B) Ingenuity Pathway Analysis of seven of the top upregulated miRNAs and their predicted targets. (C) Fold change miR-466a expression in the dLN, spleen, or mesenteric lymph node (mLN) of purified CD4⁺ cells derived from syn- or allografted mice. (D) qRT-PCR validation of mRNA expression changes in the dLN CD4⁺ cells of syn- or allografted mice; n = 8 (syngeneic) or 12 (allogeneic) mice per group. (E) Relative luciferase expression in EL-4 cells transfected with luciferase reporter constructs which contained 3′ untranslated region (3′UTR) of proteins of interest or a mutated 3′UTR, together with miR-466a-3p mimics or the negative scramble control. A total of 48 h after transfection, luciferase activity was detected. Normalized data were calculated as the quotient of Renilla/firefly luciferase activities and are presented as mean ± SEM of three independent experiments with two technical replicates indicating six measurements. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001 by Student’s t-test.