Nlrp1

Total protein was extracted from U251MG cells using RIPA buffer for 30 min on ice. The protein concentrations were detected using a BCA protein assay kit (Thermo Fisher Scientific). Total proteins (30 μg per well) were separated on 10% SDS-PAGE and then transferred onto a PVDF membrane. Following blocking with 5% nonfat milk in TBST for 1 h, the membrane was probed with primary antibodies at 4°C overnight. Later on, the membrane was incubated with the corresponding secondary antibodies (1:5000, Abcam Cambridge, MA, USA) for 1 h at room temperature. Subsequently, blots were visualized using enhanced chemiluminescence (ECL) solution (Thermo Fisher Scientific). Primary antibodies used in the assays were NLRP1 (1:1000, Abcam), cleaved caspase 1 (1:1000, Abcam), GSDMD-N (1:1000, Abcam), Pro-caspase 1 (1:1000, Abcam), GSDMD (1:1000, Abcam), and β-actin (1:1000, Abcam).

After the application of cyclic stretch, HPDLCs were washed with ice-cold PBS, scraped from the Bioflex plates and immediately lysed in a lysis buffer (Cell Signaling Technology, Danvers, MA, USA). Protein concentrations of the samples were determined by using the bicinchoninic acid protein assay kit (Pierce, Rockford, IL, USA). Protein was mixed with an appropriate volume of SDS sampling buffer and separated by SDS-PAGE gel (10%). The protein bands were then transferred onto nitrocellulose membranes by electroblotting. According to the manufacturer's instructions, the membranes were incubated with mouse monoclonal antibody against caspase-1(1:1000, Cell Signaling Technology), caspase-5 (1:1000, Cell Signaling Technology), ASC (1:1000, Santa Cruz Biotechnology, Dallas, TX, USA), NLRP1(1:1000, Abcam), NLRP3(1:1000, Abcam), GAPDH (1:10000, Cell Signaling Technology), or rabbit anti-IL-1β (1:1000, Cell Signaling Technology) primary antibodies overnight at 4°C, washed, and then incubated with anti-mouse or anti-rabbit IgG conjugated to HRP (1:5000, Cell Signaling Technology) for 60 min at room temperature. Protein bands were detected by using the ECL SuperSignal reagent (Pierce). Relative band densities of the proteins were measured from scanned films using National Institutes of Health ImageJ Software.

Val-boroPro (Talabostat mesylate; PT-100; Cat. B3941) was purchased from ApexBio (Houston, TX, USA). Agarose was purchased from Vivantis (CA, USA). N-acetyl cysteine (NAC), 3-Methyladenine (3-MA), acridine orange, and monodansylcadaverine (MDC) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). MitoTracker was from Beyotime (Shanghai, China). Rapamycin was purchased from Selleckchem (Houston, TX, USA). 3-TYP was purchased from MCE (Monmouth Junction, NJ, USA). VX-765 was purchased from AdooQ Bioscience (Irvine, CA, US). Antibodies against ATG5, Sirt3, NLRP3, caspase-1, IL-1β, MnSOD, and catalase were purchased from Cell Signaling Technology (Beverly, MA, USA). Other antibodies included NLRP1, 4-HNE, and pro-caspase-1, from Abcam (Cambridge, UK), Lamp2, from Santa Cruz Biotechnology (Santa Cruz, CA, USA), IL-18, from Affinity Biosciences Inc. (Cincinnati, OH, USA), ASC, from Omnimabs Inc. (Irvine, CA, USA), LC3B, from Sigma Chemical Co., and GAPDH, from Yeasen Biotech. (Shanghai, China). Horseradish peroxidase-conjugated secondary antibodies were purchased from ZSGB-BIO (Beijing, China).