Figure 3.

miR-24 leads to downregulation of Trb3. (A) A schematic representation of the miR-24-targeting site in the 3′-UTR Trb3 transcripts (top panel). The conserved 8-mer seed sequence is shown in grey. PASMCs were transfected with 1 nM non-specific (GFP) mimic (control) or increasing amounts (0.1, 0.3, 0.6, or 1 nM) of chemically modified, synthetic miR-24 oligonucleotides (miR-24 mimic). Twenty-four hours after transfection of mimic, cells were harvested and subjected to qRT–PCR analysis. Relative Trb3 mRNA level normalized to GAPDH (bottom left), as well as levels of mature miR-24 normalized to U6 snRNA (bottom right), is plotted as means±s.e.m. (n=3); ^*P<0.001 (versus the expression levels of control-transfected PASMCs). (B) PASMCs were transfected with 0.3 nM control mimic or 0.3 nM miR-24 mimic. Twenty-four hours after transfection of mimic, cells were stimulated with 20 ng/ml PDGF-BB for 6 h and subjected to immunoblot analysis with anti-hTrb3 antibody or anti-GAPDH antibody (loading control). The experiment was repeated three times, with similar results. (C) Cos7 cells were transfected with a construct carrying the human Trb3 cDNA construct with the 3′-UTR, which includes the miR-24 seed sequence (Trb3+3′-UTR) or deleted in the 3′-UTR (Trb3), with increasing amounts of miR-24 mimic (0.3 or 3 nM). Cells were harvested and subjected to qRT–PCR analysis (left panel) and immunoblot analysis (right panel). Relative Trb3 mRNA expression normalized to GAPDH is plotted (n=3); ^*P<0.001 (compared with no miR-24 mimic transfection (white bar) of each set). Immunoblot analysis was performed using anti-hTrb3 antibody and anti-GAPDH antibody used as loading control. The immunoblot presented is representative of three independent experiments. A full-colour version of this figure is available at The EMBO Journal Online.