Ttp488

RAGE-specific small molecule inhibitors FPS-ZM1 (4-Chloro-N-cyclohexyl-N-(phenylmethyl)benzamide; molecular weight 327.8) was purchased from MilliporeSigma (cat# 553030) and TTP488 (Azeliragon) (3-[4-[2-butyl-1-[4-(4-chlorophenoxy)phenyl]imidazol-4-yl]phenoxy]-N,N-diethylpropan-1-amine), molecular weight (532.12) was purchased from MedChemExpress (cat# HY-50682/CS-5117). Both compounds were dissolved in cell culture grade dimethyl sulfoxide (DMSO, cat# D2438, Sigma-Aldrich) and diluted with PBS to working concentration for animal studies, or with the appropriate cell culture media for in vitro studies.

Tofacitinib (S5001, Selleck, Texas, USA), fludarabine (S1491, Selleck, Texas, USA), TUNEL reaction solution (ab66108, Abcam, Cambridge, UK), TTP488 (HY-50682, MedChem Express, Sollentuna, Sweden), LPS (L8880, Solarbio, Beijing, China), caspase-1 ELISA kits (KL15200, KALANG, Shanghai, China), IL-1β ELISA kits (RLB00, R&D Systems, Minneapolis, USA), IL-18 LISA kits (ab213909, Abcam, Cambridge, UK), Anti-NLRP1 antibody (ab3683, Abcam, Cambridge, UK), anti-NeuN antibody (ab177487, Abcam, Cambridge, UK), Anti-caspase-1 antibody (BM4291, Boster, Wuhan, China), Anti-IL-1β antibody (ab9722, Abcam, Cambridge, UK), Anti-IL-18 antibody (PB0058, Boster, Wuhan, China), anti-β-actin antibody (Boster, BA2305, Wuhan, China), followed by secondary antibodies conjugated to horseradish peroxidase anti-rabbit IgG (H+L) (AS014, ABclonal, Wuhan, China), and anti-mouse IgG (H+L) (AS003, ABclonal, Wuhan, China) were used in this study.

The RAGE inhibitor TTP488 (HY-50682, MedChemExpress), the PI3K inhibitor Copanlisib (BAY 80-6946, MedChemExpress), the AKT inhibitor MK2206 (HY-108232, MedChemExpress), the mTOR inhibitor rapamycin (HY-10219, MedChemExpress), the mTOR agonist MHY1485 (HY-B0795, MedChemExpress) and the proteasome agent MG132 (HY-13259, MedChemExpress) were dissolved in DMSO as stock solutions and stored at −80 °C. The proteins were diluted to a working solution in culture medium before use. AGEs (bs-1158P, Bioss) were not dissolved in DMSO, and the stock solution was provided in liquid form. We diluted the solution to a working solution using culture medium before use. The working solution concentrations were determined by preliminary experiments, and the solution did not cause significant cell death (Supplementary Fig. 9). When the reagents were processed into working solutions, we added different amounts of DMSO depending on the concentration of the stock solution so that the concentration of DMSO in the working solution was 1/1000 (v/v). The specific concentrations are provided in Supplementary Table 1.