Fig. 3

BMAL1 participated in carbon particles/PM2.5-induced airway remodeling in vitro and in vivo. A–F After transfection with control vector or BMAL1 expression vector for 72 h, HBEs were treated with carbon particles (20 μg/ml) or PM2.5 (200 μg/ml) for 24 h or 48 h. A, B mRNA changes of collagen-I, α-SMA and MUC5AC in HBEs. n = 3. C, D Representative blot images of collagen-I and fibronectin in HBEs. E, F Representative images of immunofluorescence staining of MUC5AC in HBEs stimulated by carbon particles/PM2.5. Scale bars: 100 μm. G–J After transfection with control or specific siRNA against BMAL1 for 48 h, HBEs were incubated with 20 μg/ml carbon particles or 200 μg/ml PM2.5, after which expression levels of related mRNA and protein were measured by the RT-qPCR (24 h) and western blotting (48 h). (G, H) mRNA changes of collagen-I, α-SMA and MUC5AC in HBEs. n = 3. I, J Representative blot images of collagen-I, fibronectin and α-SMA in HBEs. Bmal1^−/− mice and Bmal1^wt/wt mice were sensitized and challenged with OVA as described in the “Methods”. Lungs of mice were harvested at day 60. K–M Representative pictures of lung tissue sections stained with Masson staining, PAS staining and immunohistochemistry staining of MUC5AC. Scale bars: 50 μm. N Collagen deposition area (%) of proximal airways in each group (n = 5). O The quantification histograms of PAS staining in proximal airways (n = 5). P The quantification histograms of IHC MUC5AC staining in proximal airways (n = 5). Data are mean ± SEM of n individual experiments. *P < 0.05, **P < 0.01, ***P < 0.001 between indicated conditions (One-way ANOVA followed by the Bonferroni’s test)