Caspase1 p20

Total protein was extracted from the right lung tissues using RIPA buffer (Meilunbio, Dalian, China). Proteins were separated by 10% or 12% SDS-PAGE and then transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, USA), which was blocked with 5% fat-free milk in TBST solution for 1 h at room temperature. The membranes were incubated with primary antibodies against LC3B (Abmart), Beclin1 (Abmart), p62 (Abmart), NF-κB p65 (Abmart), NLRP3 (Wanleibio), ASC (Cell Signaling Technology, Inc. MA, USA), pro-caspase1 (Abcam, Cambridge, UK), caspase1 p20 (Wanleibio), and mature-IL-1β (Wanleibio) at 4°C overnight. The membranes were washed with TBST and incubated with a secondary antibody (SA5-35571, Thermo Fisher, USA) at room temperature for 70 min. Immunoreactive images were detected using the Odyssey system (LI-COR Biosciences, USA) and analyzed using the ImageJ software.

Berberine Hydrochloride was purchased from National Institute for Food and Drug Control (China, Lot: 110713–201,212), dissolved in distilled water and filtered by a 0.22 μm filter. DMEM medium and Fetal Bovine Serum (FBS) were purchased from GIBCO® by Life Technologies (Carlsbad, CA, USA). The CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) (Lot: 0000185040) was purchased from Promega Corp. (Madison, WI, USA). The LDH Cytotoxicity Assay Kit (Lot: 072418181030) was acquired from Beyotime Biotechnology Co., Ltd. (Shanghai, China). The Giemsa stain solution was provided by Solarbio Life Sciences (Beijing, China). The Cytokine Magnetic Bead assay kit (#RECYTMAG-65 K) was purchased from Millipore Corp. (Billerica, MA, USA). Antibodies used in this study were P2X7 from Bioss (Beijing, China); pro-Caspase-1, ASC, NLRP3, Caspase-1 p20 and IL-18 from Wanleibio (Shenyang, Liaoning, China); IL-1β from Proteintech (Wuhan, Hubei, China); β-actin from Cell Signaling Technology (Boston, MA, USA). ReverTra Ace qPCR RT Master Mix were purchased from Toyobo CO., LTD. (Osaka, Japan). TRIzol Reagent and ABI Power SYBR Green PCR Master Mix were purchased from Thermo Fisher Scientific, China (Shanghai, China). Primers used in this study were NLRP3, Caspase-1, and IL-1β, GAPDH from iGenebio CO., LTD. (Guangzhou, Guangdong, China).