miR-378-3p targets the IL-4R/PI3K/Akt-signaling pathway. (A) Schematic depiction of the IL-4R–signaling cascade highlighting putative targets for miR-378-3p. Yellow indicates predicted by TargetScan; red: differentially expressed genes in pre- and anti-miR-378-3p–transfected fibroblasts. (B) Analysis of rIL-4–elicited STAT-6 phosphorylation by intracellular FACS staining. Histogram of STAT-6 (pY641) expression in Thio-MΦ preincubated with rIL-4 (orange line) or medium (red line) for 24 hours before restimulation with rIL-4 (open histograms) or medium (filled gray histograms) for 5 minutes. Timeline of pSTAT-6 (pY641) expression in rIL-4 (orange squares) or medium (red circles) pretreated, rIL-4–stimulated (colored symbols) or unstimulated (gray symbols) cells. Data show median fluorescence intensity of 6 individual animals ±SEM. For unstimulated controls, cells were pooled from several animals. Asterisks indicate statistical differences between rIL-4–preincubated and freshly stimulated MΦ. ***P < .001; **P < .01. (C) Luciferase-assay using the 3′UTR of wild-type Akt-1 (Akt-1) or constructs with mutated seed-region binding sites for both predicted miR-378-3p binding sites (mut) or without insert (empty). Data indicate cotransfection with a miR-378-3p mimic (black bars) or a scrambled control (open bars). Data are pooled from 5 separate experiments and depicted as relative luminescence compared with no-RNA controls (gray bars). Bars not connected by the same letters are statistically significantly different. (D) Representative Western blot analysis of RAW264.7 cells 12 hours after transfection with a mimic (pre-378) or inhibitor (anti-378) of miR-378-3p or appropriate negative controls (pre- and anti-neg). Samples were separated on 4%-12% Bis-Tris gels and stained for Akt-1 and β-actin at the same time. (E) Densitometric analysis of the samples analyzed in panel D. Data are representative of 4 separate experiments. Columns not connected by the same letters are statistically significantly different.