Pcdna3.1 nlrp3 vectors

Normal human bronchial epithelial (NHBE) cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA), and cultivated in RPMI1640 (Solarbio, Beijing, China) suspended with 10% fetal bovine serum (FBS, Solarbio, Beijing, China), 100 U/mL streptomycin (Solarbio, Beijing, China) and 100 U/mL penicillin (Solarbio, Beijing, China) at 37 °C and 5% CO₂.
NHBE cells were suspended into serum-free RPMI1640, plated into 6-well plates (1 × 10⁶ cells/mL per well), and transfected by either pCDNA3.1-NLRP3 vectors (served as the NLRP3 group) or pCDNA3.1 vectors (named the NC group) via using Lipofectamine 3000 (Thermo Fisher Scientific, Shanghai, China). The transfection was implemented following the manufacturer’s instruction. pCDNA3.1-NLRP3 vectors and pCDNA3.1 vectors were all commercially provided by GeneChem (Shanghai, China).

For the induction of the COPD cell model, NHBE cells were grown in the 6-well plates with serum-free RPMI1640 containing 5% CSE for 24 h at 37 °C and 5% CO₂ [22 (link)] (named the CSE group).
Moreover, RPMI1640 containing 10% FBS and 20 μM [19 (link)] DMF (Hengfei Biotechnology, Shanghai, China) was utilized to pretreat NHBE cells for 2 h at 37 °C and 5% CO₂. These cells then underwent incubation by serum-free RPMI1640 containing 5% CSE for 24 h 37 °C and 5% CO₂. These cells were set as the CSE + DMF group.
For NHBE cells of the CSE + DMF + NLRP3 group, they were firstly transfected by pCDNA3.1-NLRP3 vectors (GeneChem, Shnghai, China) as described in the “Cell transfection” section, then cultured for 2 h in RPMI1640 containing 10% FBS and 20 μM DMF at 37 °C and 5% CO₂, and finally incubated in serum-free RPMI1640 containing 5% CSE for 24 h at 37 °C and 5% CO₂. NHBE cells without any treatment were used as Control group. After the relevant treatment, NHBE cells of each group were harvested for Western blot analysis.