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. 2009 Dec 17;29(3):559–573. doi: 10.1038/emboj.2009.370

Figure 6.

Figure 6

miR-24 inhibits BMP signalling in vSMCs through downregulation of Trb3 (A). 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 3 nM BMP4 for 2 h and subjected to immunoblot analysis using anti-Trb3, anti-Smad1, anti-phospho-Smad1/5 (Pi-Smad1/5), or anti-GAPDH antibody (loading control). The experiment was repeated twice and produced similar results. (B) PASMCs were transfected with 0.1 nM miR-24 mimic or control mimic, followed by stimulation with 3 nM BMP4 (+BMP4) for 6 h (Id3) or for 24 h (all other markers). qRT–PCR was then performed. Relative expression of SMA, Id3, and Trb3 normalized to GAPDH and miR-24 expression normalized to U6 snRNA are plotted (means±s.e.m., n=3; *P<0.001). (C) PASMCs were transfected with 0.3 nM miR-24 mimic or control mimic, followed by adenoviral transduction of the Trb3 expression construct (Trb3 (WT) or Trb3 (ΔK)), or adenoviral transduction of GFP expression construct as control. Twenty-four hours later stimulation with 3 nM BMP4 was performed for 6 h for Id3 or 24 h for SMA. qRT–PCR was then performed and relative expression of SMA, Id3, and Trb3 normalized to GAPDH and miR-24 expression normalized to U6 snRNA are plotted (means±s.e.m., n=3; *P<0.001). (D) PASMCs were transfected with 0.3 nM control mimic or miR-24 mimic (left panel) for 24 h, followed by stimulation with 3 nM BMP4 (+BMP4) for 4 h. qRT–PCR was then performed to determine relative expression of miR-21 normalized to U6 snRNA and Trb3 expression normalized to GAPDH (means±s.e.m., n=3; *P<0.001). (E) PASMCs were transfected with 50 nM si-Control or si-Trb3 (right panel) for 24 h, followed by stimulation with 3 nM BMP4 (+BMP4) for 4 h. qRT–PCR was then performed to determine relative expression of miR-21 normalized to U6 snRNA and Trb3 expression normalized to GAPDH (means±s.e.m., n=3; *P<0.001). (F) PASMCs were transfected with 3 nM control mimic (control) or miR-24 mimic (miR-24 mimic) for 24 h, placed in starvation media for 24 h, followed by stimulation with BMP4 (+BMP4) for 48 h. Cells were trypsinized and counted using a haemocytometer. The relative number of cells is plotted means±s.e.m. (n=3); *P<0.001 (compared with unstimulated). (G) PASMCs were transfected with 3 nM control mimic (control) or miR-24 mimic (miR-24 mimic) for 24 h, followed by stimulation with BMP4 (+BMP4) for 24 h or left unstimulated (none). PASMCs were then embedded to collagen gel lattices with continued stimulation. Twenty-four hours after the collagen lattices were dissociated from the dish, gel contraction was photographed by using a digital camera (left panel). The relative size of collagen gel was quantitated using ImageJ and is plotted (means±s.e.m., n=3; *P<0.001, compared with unstimulated condition (right panel)). (H) A clone of P19 cells, stably transfected with the BMP reporter construct BRE-Luc, was transfected with 3 nM control mimic, 0.3 nM or 3 nM miR-24 mimic, or 50 nM si-Trb3, followed by 24-h BMP4 treatment. Luciferase activity was then measured and fold induction of the activity after BMP4 treatment is plotted (means±s.e.m., n=3; *P<0.001, compared with control mimic). (I) C2C12 cells were transfected with 0.003 nM control mimic (control) or 0.003 nM miR-24 mimic (miR-24) for 24 h followed by stimulation with BMP4 (+BMP4) for 2 h, or left unstimulated (none). qRT–PCR was then performed. Relative expression of ALP, Id3, and Trb3 normalized to GAPDH and miR-24 expression normalized to U6 snRNA are plotted (means±s.e.m., n=3; *P<0.001).