Lipofextamine 2000

Lentiviral expression system was deployed to manipulate the expression of RPA1 in cells. For knockdown with shRNAs, double‐strand oligos (GenePharma, Shanghai, China; sequences are provided in Table S7 in the Supporting Information) were inserted to lentiviral vector pLKO.1 following the protocols from Addgene (https://www.addgene.org/). For overexpression, full length of human RPA1 cDNA was obtained using RT‐PCR and mRNA extracted from NPC cells as template (5–8F; primer information is listed in Table S7 in the Supporting Information), and subsequently cloned into the pCDH vector (pCDH‐Flag‐RPA1); and as a negative control, the gene encoding green fluorescent protein (GFP) was also constructed into the pCDH vector separately (pCDH‐GFP). For lentivirus production, lentiviral construct was co‐transfected with packaging vectors ∆8.9 and VSVG into HEK293T cells using Lipofextamine 2000 (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. Viral supernatant was collected at 48 h post transfection and filtered through 0.45 µm PVDF filters. For transfection, lentivirus with polybrene (8 µg mL⁻¹; Sigma, San Antonio, TX, USA) were added to the cells; and subsequently the cells were maintained for 48 h, followed by selection of infected cells with 2 µg mL⁻¹ puromycin (Solarbio, Beijing, China) for one week.

ATP, Nigericin, MSU, and poly (A/T) were purchased from Sigma-Aldrich. Ultrapure LPS, Lipofextamine 2000, Pam3CSK4, MQAE, MitoSOX, MitoTracker, DAPI were acquired from Invitrogen. The Salmonella strain was a gift from Dr. Cai Zhang (Shandong University, Shandong, China). The C3 toxin was a gift from Dr. Tengchuan Jin (University of Science and Technology of China, Hefei, China). CY-09 was synthesized from Dr. Xianming Deng (Xiamen University, Xiamen, China). Protein G agarose (16-266) and anti-Flag (A2220) beads were bought from Millipore and Sigma-Aldrich, respectively. Anti-Flag (F2555) and anti-VSV (V4888) antibodies were from Sigma-Aldrich. The anti-mouse NLRP3 (AG-20B-0014) and the anti-mouse caspase-1 (AG-20B-0042) antibodies was from AdipoGen. The anti-mouse IL-1β antibody (AF-401-NA) was obtained from R&D Systems. The anti-β-actin antibody (66009-1-Ig) was from Proteintech Group. The anti-mouse ASC antibody (67824S) was from Cell Signaling Technology. The anti-mouse NEK7 antibody (ab133514) was from Abcam.