Figure 6.

Bbc3 RNAi in macrophages attenuated pro-fibrogenic effects of conditioned medium on fibroblasts. (a) MTT assay results showing that the macrophage-conditioned medium from different time points following treatment with SiO₂ induced an increase in cell viability in HPF-a cells. Data are presented as the mean±SEM; Experiments were repeated independently five times; *P<0.05; **P<0.01 versus the control group (Student's t-test). (b and c) Representative images and data showing the effects of macrophage-conditioned medium on the migration of RFP-labeled HPF-a cells. The results indicated that conditioned medium from macrophages treated with SiO₂ induced an increase in HPF-a cell migration. However, Bbc3-specific siRNA reduced the effects of macrophage-conditioned medium on HPF-a cells. Scale bar=80 μm. Quantification of the change in the scratch gap distance from six separate experiments. Data are presented as the mean±S.E.M.; **P<0.01 versus the con-siRNA group; ^##P<0.01 versus the con-siRNA+SiO₂ group (two-way ANOVA). (d) Dual immunohistochemical staining of vimentin (VIM) and COL1A2 in the lung tissue of WT and Bbc3 KO mice. Lung tissue with silicosis exhibited significantly more-intense COL1A2 expression than did control lung tissue, but the loss of BBC3 alleviated the fibrosis, as indicated by reduced COL1A2 staining intensity. Scale bar=20 μm. Images are representative of several individuals from each group (n=4). (e) Sirius Red staining results showing that Bbc3 knockout significantly alleviated pulmonary fibrosis compared with control group treated with SiO₂. Scale bar=50 μm. Images are representative of several individuals from each group (n=4)