Figure 5

The miR-19a-19b-20a sub-cluster regulates fibroblast activation in the fibrotic lung in adoptively transferred models. (A) Scheme of intratracheal-transfer experiments. Col1a2-GFP fibroblasts were transduced by retrovirus and cultured with 10% FBS + DMEM for 3 days. hCD271⁺ cells were magnetically isolated, cultured for another 10 days, and passaged three times. The harvested cells (5 × 10⁶ cells/mouse) were intratracheally transferred to the lungs of B6J mice at day 7 post-bleomycin administration, and engrafted GFP⁺ hCD271⁺ cells were recovered at day 10 by cell sorting. (B) Flow-cytometry plots of control and miR-19a-19b-20a sub-cluster fibroblasts prior to intratracheal transfer. Transduction efficiency was >98%. (C) Flow-cytometry plots of whole-lung cells from intratracheally transferred mice at day 10 post-bleomycin administration. Transduction efficiency of GFP⁺ fibroblasts was >96%. (D) The number of engrafted GFP⁺ hCD271⁺ cells at day 10 post-bleomycin administration. (E) qPCR measurement of Acta2 and Serpine1 mRNA expression in GFP⁺ hCD271⁺ cells. (D and E) Graphs show the mean ± SEM [n = 6 (control) and n = 5 (sub-cluster)]. **P < 0.01; ***P < 0.001, Student’s t-test. Cohen’s effect size d in these data was >2.7. (C,D) Representative plots [n = 5 (control) and n = 4 (sub-cluster)] are shown.