Fig. 4

BMAL1 was involved in the process of autophagy induced by carbon particles/PM2.5 in vitro and in vivo. A, B mRNA sequencing of human HBEs after knockdown BMAL1 depletion. After transfection with human BMAL1 siRNA or negative control siRNA, HBEs were incubated with carbon particles (20 µg/ml) for 24 h, after which RNA sequencing was performed. A The heatmap of differentially expressed genes, FPKM (fragments per kilobase of transcript per million mapped reads) values were represented by gradient colors and shown for each sample. Red represented a higher FPKM; blue represented a lower FPKM. Results were based on 3 RNA sequencing samples. B Pathway enrichment analysis showed differentially expressed genes that related to different pathways terms. C, D After transfection with control vector or BMAL1 overexpression vector (BMAL1 OE) for 72 h, HBEs were treated with carbon particles (20 μg/ml) or PM2.5 (200 μg/ml) for 48 h. Representative blot images of ATG7, LC3B-II and p62 in HBEs. E, F Bmal1^−/− mice and Bmal1^wt/wt mice were sensitized and challenged with OVA and carbon particles as described in the Methods. Lungs of mice were harvested at day 60. Representative pictures of lung tissue sections stained with ATG5 and LC3B. Scale bars: 50 μm