Pam2csk4

Synthetic ligands for TLR2, Pam2CSK4 and Pam3CSK4, and their biotinylated variants were purchased from Tocris Bioscience and InvivoGen. TLR9 ligand, CpG oligonucleotide 1826 (5′-tccatgacgttcctgacgtt-3′ with a phosphorothioated backbone) (CpG) and fluorescein (FAM)-labeled CpG, were commercially synthesized (Integrated DNA Technologies). Diacyl stearoyl (C18) lipid conjugated CpG (lipid-CpG) and FAM-labeled lipid-CpG were made as previously described by synthesizing diacyl C18 lipid phosphoramidite and conjugating to either CpG or CpG-FAM on a ABI 394 synthesizer on 1.0 micromole scale (11 (link)). Lipid-CpG was purified by reverse phase HPLC with a C4 column (BioBasic4, 200 mm × 4.6 mm, Thermo Scientific). A gradient eluent (Sigma-Aldrich) was implemented with 100 mM triethylamine-acetic acid buffer (pH 7.5) and acetonitrile (0–30 min, 10–100%).

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.