Additional file 1 of Investigating the impact and mechanism of Licochalcone B derivative CTG12 on NLRP3 inflammasome

Supplementary Material 1. Supplementary Figure 1. Licochalcone B derivatives inhibit NLRP3 inflammasome activation.BMDMs were primed with LPS for 4 hours, treated with Echinatin, Licorice Chalcone B, CTG4, CTG1, CTG10, CTG11, CTG12, CTG13, CTG14, CTG15, CTG16, CTG18, CTG19, CAPE, CTG23for 30 minutes, and then stimulated with nigericin for 25 minutes. Supernatants were collected for the measurement of caspase-1. Data represent as mean ± SEM. Compared to con, **** p < 0.0001; compared to a concentration of 0 μM, ###p < 0.001, #### p < 0.0001 and ns:not significant. Supplementary Figure 2. CTG11 and CTG13 inhibit NLRP3 inflammasome activation in mouse BMDMs.The structure of CTG11.Western blot analysis of IL-1β, caspase-1in culture supernatantsand pro-IL-1β, caspase-1, NLRP3, ASC in whole cell lysatesof LPS-primed BMDMs treated with CTG11 and then stimulated with Nigericin, supernatants were collected for the measurement of caspase-1, IL-1β, LDHand TNF-α.The structure of CTG13.Western blot analysis of IL-1β, caspase-1in culture supernatantsand pro-IL-1β, caspase-1, NLRP3, ASC in whole cell lysatesof LPS-primed BMDMs treated with CTG13 and then stimulated with Nigericin, supernatants were collected for the measurement of caspase-1, IL-1β, LDHand TNF-α. Coomassie blue–stained gels used as loading control and Lamin B used as a control for equal loading of the samples. Data represent as mean ± SEM. Compared to con, ** p < 0.01, ***p < 0.001, **** p < 0.0001; compared to a concentration of 0 μM, ###p < 0.001, ####p < 0.0001 and ns: not significant. Supplementary Figure 3. CTG12 impedes the priming process of NLRP3 inflammasome activation and specifically inhibits canonical and noncanonical NLRP3 inflammasome activation.BMDMs were primed with LPS treated with CTG12, then stimulated with Nigericin ATP, poly, or SiO₂. Supernatants were collected for the measurement of TNF-α, BMDMs primed with Pam3CSK4 treated with CTG12, followed by cytosolic LPS. Supernatants were collected for the measurement of TNF-α, BMDMs were primed with LPS treated with CTG12, then stimulated with Nigericin, salmonella, poly. Supernatants were collected for the measurement of TNF-α.THP1 were pretreated for 1 h with various concentrations of CTG12 and then transfected with poly. P-IRF3, STING, IRF3, and Lamin B were analyzed by western blotting 2 h after polytransfection. The expression of IFN-β, TNF-α, CXCL10mRNA was detected by qPCR assay 4 h after polytransfection.BMDMs were cultured with LPS for 4 h, then incubated with CTG12 for 1 h, or BMDMs were first treated with CTG12 for 1 h, followed by stimulating with LPS for 4 h, quantitative PCR was used to detect IL-6and TNF-αmRNA. Data represent as mean ± SEM. Compared to con, *** p< 0.001, **** p < 0.0001; compared to a concentration of 0 μM, # p< 0.05, ###p < 0.001, #### p < 0.0001 and ns: not significant