BEAS-2B cell culture and treatment

A human bronchial epithelial cell line BEAS-2B (American Type Culture Collection, Manassas, VA, USA) was cultured in hormonally defined Ham’s F12 medium (HD-F12) supplemented with 1% penicillin-streptomycin, 5 μg/ml insulin and transferrin, 25 ng/ml EGF, 15 μg/ml endothelial cell growth supplement (Collaborative Research, Inc., Bedford, MA, USA), 200 pM triiodothyronine (Gibco, Carlsbad, CA, USA), and 1 mM hydrocortisone.

To investigate whether DEP induces bronchial epithelial cell inflammation via autophagy, BEAS-2B cells were pre-treated with 1.5 mM 3-MA (Sigma-Aldrich) or 10 nM Rap (Sigma-Aldrich) for 1 h, and then exposed to 5 ppm SRM 1650b for 12 h. Western blot, IF, and ELISA were then applied to detect the autophagy-related proteins LC3B and p62 expression, and pro-inflammatory cytokines TNF-a, IL-6, and IL-8 level in culture supernatants.

To investigate whether CC16 secretion from CC16⁺ cells protects against DEP-induced bronchial epithelial cell inflammation, BEAS-2B cells were divided into four groups and cultured with different medium for 12 h, including ctrl group (equiproportional mixing of fresh DMEM/F12 and HD-F12), DEP group (the same medium with ctrl group but containing 5 ppm SRM 1650b), DEP+CC16⁺ group (CC16⁺ medium containing 5 ppm SRM 1650b), and DEP+DEP-CC16⁺ group (DEP-CC16⁺ medium containing 5 ppm SRM 1650b). Next, the expression of p62 and LC3B protein, and the levels of TNF-a, IL-6, and IL-8 level were determined by western blot, IF, and ELISA, respectively.

For further confirmation the protection role of CC16 against DEP-induced bronchial epithelial cell inflammation, BEAS-2B cells were exposed to 5 ppm SRM 1650b in the presence of rCC16 (prepared by our laboratory) or not for 12 h. Then autophagy and inflammation were determined.