Tlrlmdp

To electroporate LPS or MDP, the Neon Transfection System (MPK5000, Invitrogen) and the Neon Transfection System 100 μL Kit (MPK10025, Invitrogen) were used. Per each tip, 5 ×10^5 cells were transfected with the indicated amount of ultrapure lipopolysaccharide (LPS) from E. coli 111:B4 (tlrl-3pelps, Invivogen) or MDP (tlrlmdp, Tocris). Electroporation parameters were set at 1500 V, 30 ms and pulse number 1 for IMR90 cells and 1100 V, 20 ms and pulse number 2 for HEK293T cells. Once electroporated, the tip content was unloaded into a clean Eppendorf tube and tubes were centrifuged on a bench-top centrifuge at 3000 rpm 3 min. The supernatant was removed to avoid any traces of MDP or LPS in the extracellular media prior to plating. For CAPAN-1, PSN-1, A549, and HCT116, 1 to 10 μg/mL MDP and LPS were transfected in DMEM media with 10% FugeneHD (Promega).

OIS was induced by treating IMR90 ER:RAS cells with 100 nM 4OHT (H7904, Sigma). IMR90 ER:RAS and control ER:STOP were maintained in standard media supplemented with 200 μg/ml geneticin (10131-027, ThermoFisher). To induce oncogene-induced senescence, IMR90 ER:RAS cells were treated with 100 nM 4-hydroxytamoxifen (4OHT) for 48 h, followed by 5 days in normal culture media, For DNA damage-induced senescence, IMR90 cells were treated with 100 μM Etoposide for 48 h. For noncanonical inflammasome activation and inflammasome priming experiments, ultrapure lipopolysaccharide (LPS) from E. coli 111:B4 (tlrl-3pelps, Invivogen), muramyldipeptide (MDP) (tlrlmdp, Tocris), Pam2CSK4 (4637, Tocris), Pam3CSK4 (4633, Tocris), Recombinant Human Apo-SAA (A-SAA)(300-13, Peprotech) and BSA (Sigma) were used. For priming time-course experiments, the following concentrations were used: LPS (1 μg/ml), MDP (1  μg/ml), Pam2CSK4 (50 ng/ml), Pam3CSK4 (500 ng/ml). To prime inflammasomes prior to LPS transfection, cells were treated with Pam2CSK4 (1 μg/ml) A-SAA (10 μg/ml) or BSA (10 μg/ml) for 3 h. Ouabain (Apexbio) (100 nM) was used as a senolytic.