Figure 4

The miR-19a-19b-20a sub-cluster inhibits activation of primary lung fibroblasts in vitro. (A–C) Primary lung fibroblasts transduced by retrovirus were cultured with 1% FBS + DMEM for 12 h, followed by a change of medium to 1% FBS + DMEM in the presence or absence of TGF-β1. (A) Effect of the miR-19a-19b-20a sub-cluster on TGF-β1-mediated expression of Acta2 mRNA in primary lung fibroblasts. qPCR measurement of mRNA expression after a 36-h stimulation with TGF-β1 (10 ng/mL). (B) Effect of the miR-19a-19b-20a sub-cluster on Acta2- and Col1a2-promoter activity in Acta2-Kusabira Orange2 × Col1a2-GFP fibroblasts under TGF-β1-stimulated conditions. Flow-cytometric analysis of the MFIs associated with Acta2-Kusabira Orange2 and Col1a2-GFP fibroblasts in hCD271⁺ cells after a 36-h stimulation with TGF-β1 (10 ng/mL). (C) Fluorescent images of Acta2-Kusabira Orange2 × Col1a2-GFP fibroblasts after a 36-h stimulation with TGF-β1 (10 ng/mL). Acta2-Kusabira Orange2 (magenta) and Col1a2-GFP (green). Scale bars: 100 μm. Representative images (n = 3) are shown. (D,E) Effect of the miR-19a-19b-20a sub-cluster in the contractile activity of primary lung fibroblasts in the presence or absence of TGF-β1 stimulation. Col1a2-GFP fibroblasts (3 × 10⁴) transduced by retrovirus were plated on collagen gels. After 2 h, the gels were mechanically loosened from the sides of the wells, and the cells were cultured in medium containing TGF-β1 (10 ng/mL) and 1% FBS + DMEM. The surface area of the collagen gel was measured using ImageJ at 0- and 7-h post-stimulation, and the percentage of gel contraction in each cell group was calculated. (D) Gel images at 7-h post-stimulation. Dashed line shows the size of the gels at the stimulation-start point. Representative images (n = 3) are shown. (E) Percentage of collagen gel contraction at 7-h post-stimulation. Graphs show the mean ± SEM (n = 7; A), (n = 6; B), and (n = 3; E). *P < 0.05; **P < 0.01; ***P < 0.001, Student’s t-test (A,B, and E). Cohen’s effect size d in these data was >1.8.