Anti zo 1

The total protein of lung tissues and cells was collected with RIPA buffer (Servicebio, Wuhan, China) with cocktail and phosphatase inhibitors (Thermo Fisher Scientific, Wuhan, China), followed by electrophoresed through 8–12% sodium dodecyl sulfate–polyacrylamide gel and then transferred to 0.45 μm PVDF membrane (Merck Millipore, USA). A total of 30 ug of protein was used for western blot experiments. After blocked with 5% milk, the membrane was immunoblotted with following primary antibodies(Except for labeled, all from Cell Signaling Technology, MA, USA) at 4 °C overnight: anti-ERRα (1:1000), anti-ZO-1 (1:1000; Abcam), anti-VE-cadherin (1:1000), anti-Occludin(1:1000), anti-JAM-A(1:1000; Abcam), anti-Sirt3 (1:1000), anti-Bcl-2 (1:1000), anti-Bax (1:1000), anti-Smac (1:1000), anti-Cytochrome c (1:1000), anti-caspase 3 (1:1000), anti-cleaved caspase 3 (1:500), anti-caspase 9 (1:1000), anti-cleaved caspase 9 (1:500), anti-LC3A/B(1:1000), anti-Beclin1 (1:1000), anti-p62 (1:1000), or anti-GAPDH (1:10000, Abcam, internal control). After incubated with the HRP-conjugated secondary antibodies at 25℃ for 1 h, the immunoblots were visualized using the ECL system. The gray value was analyzed using the Image J and calculate the relative protein level based on the density ratio of the target protein to GAPDH (internal control).

Microfuge 22R desktop refrigerated centrifuge (Beckman, Germany), ultra-low temperature refrigerator (Anhui Zhongke Duling Co., Ltd.), electric thermostatic water bath (Changzhou Putian Instrument Manufacturing Co., Ltd.), electronic analytical balance [Mettler-Toledo Instruments (Shanghai) Co., Ltd.], electric heating constant temperature blast drying oven (Shanghai Yuejin Medical Equipment Factory), physiological saline (Shijiazhuang Siyao Co., Ltd.), BSA24S-CW electronic analytical balance (Sartorius, Germany), paraformaldehyde fixative solution (Wuhan Sewell Biotechnology Co., Ltd.); TissueLyser-24 (Shanghai Jingxin Industrial Development Co., Ltd.), BCA protein concentration assay kit (Beijing Solarbio company), NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (ThermoFisher Scientific, United States), Mouse IFN-γ enzyme-linked immunosorbent assay kit (Jianglai Biotechnology), Mouse TNF-α ELISA kit (Jianglai Biotechnology), Mouse IL-6 ELISA kit (Jianglai Biotechnology), Anti-Claudin 1 (Abcam, United States); Anti-Occludin (Abcam, United States), Anti-ZO1 (Abcam, United States); Anti-HIF-1α (Affinity Biosciences, United States); Anti-NF-κB p65 (Abcam, United States); Anti-STAT1 (Proteintech).

Anti-ZO-1, anti-E-Cadherin, anti-TLR-4, and anti-NF-κB antibodies were purchased from Abcam (Cambridge Science Park in Cambridge, UK). TNF-α, IL-1β, and IL-6 enzyme-linked immunosorbent assay (ELISA) kits were obtained from Expandbiotech (Life Science Park, Beijing, China). Real-time quantitative PCR kit LightCycler® 480 SYBR Green I Master (cat. no. 04707516001, USA) was purchased from Roche Applied Science (Basel, Swiss).
Real-time quantitative PCR instrument (Science 384, Applied Basel, Swiss), microplate reader (Multiskcan FC, Applied Thermo Fisher, USA), and electrophoresis apparatus (PowerPac Basic, Applied Bio-Rad, USA) were used in the study.

After washing the cells with PBS, our team lysed them with RIPA lysis buffer (Thermo Science) containing trypsin inhibitor aprotinin, collected supernatant after high-speed centrifugation, heated it in water bath to denature its protein, quantified protein through BCA, separated protein via SDS-PAGE gel electrophoresis, transferred it to a nitrocellulose membrane (Millipore), later sealed the membrane with skimmed milk powder for 30 minutes at 37°C with 5% CO₂, added primary antibody and Anti-GAPDH (1:3000, Abcam, ab8245) for incubation overnight at 4°C, and applied primary antibodies Anti-KAT2B (1:1000, Abcam, ab176316), Anti-E-cadherin (1:1000, Abcam, ab1416), Anti-N-cadherin (1:1000, Abcam, ab202030), Anti-Vimentin (1:1000, Abcam, ab193555) as well as Anti-ZO-1 (1: 20,000, Abcam, ab182159). After rinsing NC membrane with TBST solution, our team incubated it with a secondary antibody (Hubei Bios Biotech Co., Ltd., 1:2000) at room temperature for 1 h, and then developed it using ECL (CST), with GAPDH as an internal reference.

IL-1β, IL-18, ZO-1 and Occludin in the lung and small intestine were identified by immunofluorescence using primary antibodies (1:300 dilution) including anti-IL-1β (cat. no. ab9722, Abcam), anti-IL-18(cat. no. ab71495, Abcam), anti-ZO-1(cat. no. ab216880, Abcam) and anti-occludin (cat. no. ab222691, Abcam). After washing, the slides were incubated with secondary secondary antibody for 1 h at room temperature in the dark. Then the lung and small intestine tissues were stained with 40, 6-diamidino-2-phenylindole (DAPI) and observed with a fluorescence microscope.