Figure 7. PIM1 inhibition inhibits profibrotic gene expression and collagen secretion in organotypic IPF lung cultures ex vivo.

(A) Schematic of organotypic culture of IPF lungs. (B) H&E and trichrome staining of IPF lung tissue explants after 5 days in culture showing intact tissue architecture of nonfibrotic area (top panels) and distorted architecture of fibrotic area (bottom panels). Scale bar: 50 μm. FF, fibrotic foci; V, vein; Av, alveoli. (C) qPCR analysis of ECM gene expression in IPF lung explants treated with 10 μM of AZD1208 or DMSO control in combination with or without 10 ng/mL of TGF-β for 5 days. n ≥ 3 IPF lung explants. Data are shown as mean ± SEM. P values were calculated using 1-way ANOVA with Holm-Šidák post hoc test. (D) qPCR analysis of pathogenic lung fibroblasts gene markers in IPF lung explants treated with 10 μM of AZD1208 or DMSO in the presence or absence of 10 ng/mL of TGF-β for 5 days. n ≥ 3 IPF lung explants. Data are shown as mean ± SEM. P values were calculated using 1-way ANOVA with Holm-Šidák post hoc test. (E) qPCR analysis of prosurvival genes in IPF lung explants treated with 10 μM of AZD1208 or DMSO in the presence or absence of 10 ng/mL of TGF-β for 5 days. n = 3 IPF lung explants. Data are shown as mean ± SEM. P values were calculated using 1-way ANOVA with Holm-Šidák post hoc test. (F) Soluble collagen-I secreted from IPF lung explants into the media was evaluated by Western blot analysis. Each lane contained equal volume of conditioned medium of different lung sections obtained from single IPF lung explants. Ponceau staining was used as loading control for secreted collagen-I.