Figure 3. Treatment of lung organ cultures with anti-miRs and mimics alters airway branching.

E14 mouse fetal lungs were harvested and cultured on porous membranes for 72 hours. (A) Lungs were transfected the day after harvest with anti-SCR, anti-221 or anti-130a. Downregulation of miR-221 (blue bar) and miR-130a (red bar) was confirmed by qRT-PCR. Mean ± SEM of N = 3 experiments. (B) Brightfield images were taken after 24 hours of treatment. After 48 hours of treatment, lungs were fixed and stained for E-cadherin. Z-stack confocal images of whole mount E-cadherin stained lungs were obtained and the final image of the entire lung obtained by software combination of the z-stacks. Altered airway branching is seen after 24 and 48 hours after transfection. Anti-221 lungs have increased proximal and distal airway branching, and narrower terminal branch tips. Anti-130a lungs have wider and fewer airway branches. (C) Width of distal airway tips was measured for each condition and compared to Anti-SCR. Mean ± SEM of N ≥3, *p<0.01. (D) Lungs were transfected the day after harvest with SCR, Mimic 221 or Mimic 130a. Upregulation of miR-221 (blue bar) and miR-130a (red bar) was confirmed by qRT-PCR. Mean ± SEM of N = 3 experiments. (E) Brightfield images were taken after 24 hours of treatment. After 48 hours of treatment, lungs were fixed and stained for E-cadherin. Z-stack confocal images of whole mount E-cadherin stained lungs were obtained and the final image of the entire lung obtained by software combination of the z-stacks. Mimic 221 lungs had decreased airway branching whereas mimic 130a had increased central and peripheral branching. (F) Width of distal airway tips were measured for each condition and compared to SCR. Mean ± SEM of N ≥3.