Casp9

ExoQuick-TC exosome-isolation reagent (EXOTC50A-1), SeraMir exosome RNA-isolation kit (RA808A-1), exosome-specific marker anti-αCD63 antibody (EXOABCD63A-1), and florescence exosome-preparation reagent (EXDC300A-1) were from System Biosciences (SBI, Palo Alto, CA, USA). Penicillin-streptomycin (PS) solution, trypsin-EDTA solution, RPMI medium, and α-MEM media were obtained from Life Technologies GIBCO (Grand Island, NY, USA). Fetal bovine serum (FBS) was from Sigma-Aldrich (St. Louis, MO, USA). Antibodies used for immunoblotting were against phosphorylated p53 (Cell Signaling Technology, Beverly, MA, USA), BAX, BCL2, CASP9, CASP3, β-actin (Abcam, Cambridge, MA), and cytochrome c (ENZO Diagnostics Inc., Farmingdale, NY, USA).

Tissue lysates were homogenized in SDS lysis buffer [Cell Signaling Technology (CST), #7722] and transferred onto polyvinylidene difluoride membranes. After blocking in 5% milk in tris-buffered saline and 0.1% Tween 20, membranes were incubated overnight with the following antibodies at 4°C: CASP9 (Abcam, #ab202068), cleaved CASP3 (CST, #9664), RIP3 (Millipore Sigma, #PRS2283), BAX (CST, #2772), BCL2 (CST, #3498), GSDMD (Santa Cruz Biotechnology, sc-393656), NLRP3 (CST, #15101), ACSL4 (Abcam, #ab155282), LC-3 (CST, #2775S), cGAS (CST, #31659), STING (CST, #13647S), TBK1 (CST, #38066), pTBK1 (CST, #5483), IRF3 (CST, #4302), pIRF3 (CST, #4947), p65 (CST, #8242), pp65 (CST, #3033), and GAPDH (CST, #5174). Membranes were incubated with horseradish peroxidase–conjugated secondary anti-rabbit or anti-mouse antibody (CST, #7074 and #7076) for 1 hour at room temperature. Signal was detected by enhanced chemiluminescence (SuperSignal West Femto Maximum Sensitivity Substrate, #34094; Thermo Fisher Scientific). Western blots were quantified using the Fiji software (58 (link)).

Reagent and kits were derived from different commercial companies, such as a Small-RNA isolation kit (RA808A-1; SBI, Palo Alto, CA, USA), Mir-X™ miRNA First Strand Synthesis and SYBR® qRT-PCR kit (Cat. No. 638316; Clontech Laboratories, Inc., Mountain View, CA, USA), Dual-Luciferase® Reporter Assay System (Cat. No. E1910; Promega Corporation, Madison, WI, USA) and Dulbecco’s modified Eagle’s medium (DMEM)-high glucose medium and phosphate buffered saline (PBS; Hyclone Laboratories, Inc. UT, USA). Roswell Park Memorial Institute (RPMI) medium, penicillin-streptomycin (PS) solution, and trypsin-EDTA (TE) solution were obtained from Life Technologies GIBCO (Grand Island, NY, USA). Antigen probing primary antibodies used for immunoblotting against BCL2 (Santa Cruz, CA, USA), CASP9, PARP, and β-actin (Abcam, Cambridge, MA, USA). Fetal bovine serum (FBS) and other analytical grade reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA), unless otherwise stated.