FIEL1 knockdown ameliorates bleomycin-induced lung injury in vivo. (A) C57BL/6J mice were treated i.t. with Lenti-CON shRNA or Lenti-FIEL1 shRNA (107 PFU/mouse) for 144 h; mice were then treated i.t. with bleomycin (0.05 U). Mice were euthanized over the next 1–21 d, and lungs were lavaged with saline, harvested, and then homogenized. (B–D) Lavage protein, total cells, and CXCL1 concentrations were measured. Data represent mean values ± SEM (n = 4–6 mice per group, data are from one of the two experiments performed; *, P < 0.05 compared with Control, Student’s t test). (E–G) Lavage cells were then processed for Wright-Giemsa stain; lavage macrophages, neutrophils, and lymphocytes were counted and graphed. Data represent mean values ± SEM (n = 4–6 mice per group, *, P < 0.05 compared with Control, Student’s t test). (H) Survival studies of mice that were given bleomycin. Mice were carefully monitored over time; moribund, preterminal animals were immediately euthanized and recorded as deceased. Kaplan-Meier survival curves were generated using SPSS software (n = 8 mice per group; *, P < 0.05 compared with Control, Log-rank test P < 0.05). Empty: n = 8, FIEL1: n = 8. (I and J) H&E and Trichrome staining was performed on lung samples. Bar indicates 100 µm. (K) Collagen percentage quantification from Trichrome staining. Data represent mean values ± SEM (n = 4–6 mice per group, *, P < 0.05 compared with Control, Student’s t test). (L) Hydroxyproline content was measured in lungs from 7, 14, and 21 d after bleomycin challenge. Data represent mean values ± SEM (n = 4–6 mice per group; *, P < 0.05 compared with Control, Student’s t test). (M) Mice lungs were isolated and assayed for PIAS4 and FIEL1 immunoblotting.
