Figure 2.

Deletion of TAK1 in lung tissues by lentiviral-based CRISPR/Cas9 system reduced inflammation and fibrosis in silica-exposed mice. (a) A schematic diagram illustrating the experimental design. Before induction of inflammation and fibrosis in lungs by silica exposure, the mice were infected intratracheally with lentiviral vector expressing CRISPR/Cas9 system (sgTAK1–3 and Cas9) or lentiviral vector control. (b) ELISA to determine silica exposure-induced levels of IL-1β (left), TGF-β (middle) and TNF-α (right) in the supernatant of BALF from age-matched mice or mice infected with specified lentiviral vectors. (c) Gelatin zymography (upper: representative images; bottom: relative bands intensity) to examine silica exposure-induced activities of matrix metalloproteinases (MMP-9 and MMP-2) in homogenized lung tissues from age-matched mice or mice infected with specified lentiviral vectors. (d) MMP-targeted NIR fluorescence imaging showing silica exposure-induced activities of matrix metalloproteinases in lung tissues from age-matched mice or mice infected with specified lentiviral vectors. Scale bar, 1 cm. (e) Western blotting (left: representative images; right: relative bands intensity) to examine silica exposure-induced inflammation-related downstream signaling in primary alveolar macrophages isolated from lung tissues of age-matched mice or mice infected with specified lentiviral vectors. (f) Hydroxyproline assay to determine silica exposure-induced total collagen levels in lung tissues from age-matched mice or mice infected with specified lentiviral vectors. (g) Western blotting (upper: representative images; bottom: relative bands intensity) to determine silica exposure-induced levels of Col I and Col III in lung tissue from age-matched mice or mice infected with specified lentiviral vectors. (h) Histological examination to determine silica exposure-induced fibrotic nodule formation (arrows indicated in H&E staining) and collagen deposition (arrows indicated in immunohistochemical staining) in lung tissue from age-matched mice or mice infected with specified lentiviral vectors. Scale bars, 50 μm. (i) Western blotting (left: representative images; right: relative bands intensity) to examine silica exposure-induced fibrosis-related downstream signaling in primary fibroblasts isolated from lung tissues of age-matched mice or mice infected with specified lentiviral vectors. Data are presented as mean±s.d. *P<0.05, n=9 per group. One-way ANOVA with a post hoc test was performed and the statistical differences between the two groups were determined by the Student’s t-test.