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.
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.