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

补充材料1。
补充图1：甘草查尔酮B（Licochalcone B）衍生物抑制NLRP3炎症小体（NLRP3 inflammasome）活化。将骨髓来源巨噬细胞（bone marrow-derived macrophages, BMDMs）用脂多糖（lipopolysaccharide, LPS）致敏预处理4小时，分别用紫铆因（Echinatin）、甘草查尔酮B、CTG4、CTG1、CTG10、CTG11、CTG12、CTG13、CTG14、CTG15、CTG16、CTG18、CTG19、咖啡酸苯乙酯（CAPE）、CTG23处理30分钟，随后用尼日利亚菌素（nigericin）刺激25分钟。收集细胞上清液用以检测半胱天冬酶-1（caspase-1）的表达。实验数据以平均值±标准误（mean ± SEM）表示。与对照组（con）相比：**** p < 0.0001；与0 μM浓度组相比：### p < 0.001、#### p < 0.0001，ns表示无显著性差异（not significant）。
补充图2：CTG11与CTG13可抑制小鼠骨髓来源巨噬细胞中的NLRP3炎症小体活化。
首先展示CTG11的分子结构。对经脂多糖致敏预处理、并用CTG11处理后再经尼日利亚菌素刺激的骨髓来源巨噬细胞，采用蛋白质印迹法检测细胞培养上清液中的白细胞介素-1β（IL-1β）、半胱天冬酶-1，以及全细胞裂解液中的前体白细胞介素-1β（pro-IL-1β）、半胱天冬酶-1、NLRP3、凋亡相关斑点样蛋白（ASC）；收集细胞上清液用以检测半胱天冬酶-1、IL-1β、乳酸脱氢酶（LDH）与肿瘤坏死因子-α（TNF-α）。
随后展示CTG13的分子结构。对经脂多糖致敏预处理、并用CTG13处理后再经尼日利亚菌素刺激的骨髓来源巨噬细胞，采用蛋白质印迹法检测细胞培养上清液中的IL-1β、半胱天冬酶-1，以及全细胞裂解液中的pro-IL-1β、半胱天冬酶-1、NLRP3、ASC；收集细胞上清液用以检测半胱天冬酶-1、IL-1β、LDH与TNF-α。本实验采用考马斯亮蓝染色凝胶作为上样对照，核纤层蛋白B（Lamin B）作为样本上样量均一性的内参蛋白。实验数据以平均值±标准误表示。与对照组相比：** p < 0.01、*** p < 0.001、**** p < 0.0001；与0 μM浓度组相比：### p < 0.001、#### p < 0.0001，ns表示无显著性差异。
补充图3：CTG12可阻断NLRP3炎症小体活化的启动过程，并特异性抑制经典与非经典NLRP3炎症小体活化。
将骨髓来源巨噬细胞用脂多糖致敏预处理后，用CTG12处理，随后分别用尼日利亚菌素、三磷酸腺苷（ATP）、聚肌苷酸-聚胞苷酸（poly）或二氧化硅（SiO₂）刺激，收集细胞上清液用以检测TNF-α。
将骨髓来源巨噬细胞用Pam3CSK4预处理后用CTG12处理，随后转染胞质脂多糖，收集细胞上清液用以检测TNF-α。
将骨髓来源巨噬细胞用脂多糖致敏预处理后用CTG12处理，随后分别用尼日利亚菌素、沙门氏菌（salmonella）、聚肌苷酸-聚胞苷酸刺激，收集细胞上清液用以检测TNF-α。
将THP1细胞用不同浓度的CTG12预处理1小时，随后转染poly。于poly转染2小时后，采用蛋白质印迹法检测P-IRF3、干扰素基因刺激蛋白（STING）、干扰素调节因子3（IRF3）与Lamin B的表达；于poly转染4小时后，采用实时定量聚合酶链反应（qPCR）检测干扰素-β（IFN-β）、TNF-α、趋化因子配体10（CXCL10）的mRNA表达水平。
将骨髓来源巨噬细胞与脂多糖共培养4小时，随后加入CTG12孵育1小时；或先用CTG12处理骨髓来源巨噬细胞1小时，再用脂多糖刺激4小时，随后采用qPCR检测白细胞介素-6（IL-6）与TNF-α的mRNA表达水平。
实验数据以平均值±标准误表示。与对照组相比：*** p < 0.001、**** p < 0.0001；与0 μM浓度组相比：# p < 0.05、### p < 0.001、#### p < 0.0001，ns表示无显著性差异。