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. 2011 Dec 9;287(6):3976–3986. doi: 10.1074/jbc.M111.303156

FIGURE 5.

FIGURE 5.

miR-21-mediated inhibition of DOCKs modulates vSMC motility. A, PASMCs were transfected with control cel-miR-67 mimic or miR-21 mimic. Active GTP-bound Rac1 was pulled down by GST-Pak1-PBD fusion protein and visualized by immunoblotting with anti-Rac1 antibody (top panel, Rac1-GTP). Immunoblotting of total cell lysates with anti-Rac1 antibody is shown to demonstrate total amount of Rac1 (top panel, Total Rac1). The level of Rac1-GTP relative to total Rac1 is presented as a relative Rac1 activity (bottom panel). B, PASMCs transfected with control cel-miR-67 mimic (Control; black diamonds), miR-21 mimic (miR-21; red circles), or anti-miR-21 (magenta triangles) were subjected to the scratch wound assay. As a control, PASMCs were treated with 3 nm BMP4 (green squares). The distance of migration was measured using ImageJ over 9 h after a scratch wound was introduced. C, PASMCs transfected with 25 nm non-targeting control siRNA (siControl); siRNA against DOCK4 (siDOCK4), DOCK5 (siDOCK5), or DOCK7 (siDOCK7); or miR-21 mimic (miR-21) were subjected to the scratch wound assay. D, PASMCs transfected with siControl or siDOCK4 were subjected to the scratch wound migration assay in the presence or absence of 50 μm Rac1 inhibitor (NSC23766). E, rat PASMC PAC1 cells were transfected with control vector (Control) or DOCK7 expression construct carrying the DOCK7 cDNA deleted in the 3′-UTR (DOCK7wo3′-UTR) followed by the scratch wound assay in the presence or absence of 3 nm BMP4. F, levels of DOCK4, -5, or -7 mRNA relative to GAPDH or miR-21 relative to U6 snRNA were measured by qRT-PCR in PASMCs stimulated with 20 ng/ml PDGF-BB or 3 nm BMP4 for 24 h. Data represent mean ± S.E. *, p < 0.0001; **, p < 0.01; ***, p < 0.001. G, PASMCs transfected with siRNA (siControl, siDOCK4, siDOCK5, or siDOCK7) were subjected to the scratch wound assay in the presence or absence of 20 ng/ml PDGF-BB. Data represent mean ± S.E. H, PASMCs were transfected with siControl, siDOCK4, siDOCK5, siDOCK7, miR-21 mimic, or anti-miR-21 followed by stimulation with 3 nm BMP4 or no stimulation (None) for 24 h. The PASMC embedded collagen lattices were photographed by using a digital camera to measure gel contraction. The relative size of collagen gel was quantitated using ImageJ. Data represent the mean percentage of the collagen gel shrinkage compared with unstimulated control. I, levels of contractile genes. α-Smooth muscle actin (ASMA) and calponin1 (CNN) mRNA levels relative to GAPDH were measured by qRT-PCR in PASMCs transfected with siControl (siCont), siDOCK4, siDOCK5, or siDOCK7. Data represent mean ± S.E. *, p < 0.01.