Penicillin and streptomycin

Tanreqing injection (TRQ, 33 mg/ml) was provided by Shanghai Kaibao Pharmaceutical Company, China, Lot. No. 2003210. LPS (Escherichia coli 055: B5, L2880) was purchased from Sigma-Aldrich (St. Louis, MO, United States). Fetal bovine serum (FBS) was obtained from Invitrogen Gibco (Grand Island, NY). Penicillin and streptomycin, Dulbecco’s modified Eagle’s medium (DMEM), 0.25% trypsin, and phosphate buffer saline (PBS) were purchased from Meilunbio (Dalian, China). Antibodies against cGAS, P-STING, STING, P-TBK, TBK, P-IRF3, IRF3, NF-κB p65, P-P65, and P-IκBα were obtained from Cell Signaling Technology (Beverly, MA, United States). All the immunosorbent assay (ELISA) used in this study were acquired from Multiscience (Zhejiang, China), Elabscience (Wuhan, China), and Neobioscience Technology Company (Shenzhen, China). The MPO, LDH, MDA, GSH, and SOD assay kits were purchased from Nanjing Jiancheng Biology Institution (Nanjing, China). DNeasy Blood & Tissue Kit was purchased from Qiagen (Hilden, Germany). TB Green Premix Ex Taq II was acquired from TaKaRa (Beijing, China).

Mouse primary adipocytes, 3T3-L1, and mouse myoblasts, C2C12, were purchased from the Kunming Cell Bank of the Chinese Academy of Sciences (Kunming, China). Cells were maintained in DMEM (Meilunbio, Dalian, China) supplemented with 10% FBS (Biological Industries, Beit Haemek, Israel) and 100 U/mL penicillin and streptomycin (Meilunbio) in a CO₂ incubator (5% CO₂) at 37 °C until confluence.
Prior to the experiment, 3T3-L1 cells were cultured and differentiated into adipocytes. After full confluence, cells were incubated with DMEM containing 10% FBS and MDI (IBMX 0.5 mM, DEX 1 μM, and insulin (INS, 5 μg/mL)) for 72 h. The medium was replaced with 10% FBS and insulin (INS, 5 μg/mL) in DMEM for 72 h, followed by DMEM containing 10% FBS every 2 days until differentiation was complete. C2C12 cells were cultured and differentiated into muscle cells. Fully confluent cells were further cultured with DMEM containing 2% HS (Gibco, Carlsbad, CA, USA) for 7 days, with changes every 2 days until differentiation was complete. Prior to drug treatment, cells were starved in serum-free medium for 4 h and treated with insulin (1 nM or 100 nM) (Novo Nordisk, Copenhagen, Switzerland) and serpentine (10 μM) alone, or in combination, for 20 min for further experiments. Serpentine was purchased from Yunnan XiLi Biotechnology Co., LTD (BioBioPha, Kunming, China).

MCF-7 and HCT-116 cells were purchased from the Meilunbio Biotechnology Co., Ltd. (Dalian, China). Cells were cultured at 37 °C with 5% CO₂, using DMEM or RPMI 1640 medium (Meilunbio, Dalian, China) with 10% (v/v) fetal bovine serum (Gibco, San Diego, CA, USA) and 1% (v/v) penicillin and streptomycin (Meilunbio, Dalian, China). CCK-8 assay was used to determine the inhibitory effect in vitro. In short, cells were incubated into 96-well plates at the rate of 5000 cells per well. After 24 h of incubation, 100 μL medium was added, which contained a specific concentration of the test compound. After 72 h, 10 μL CCK-8 regent was added and the plate was incubated at 37 °C, 5% CO₂ for 1 h. The optical density of each hole at 450 nm was read by a BioTek TS-800 microplate reader. GraphPad Prism 9.3.1 software was used to calculate the IC₅₀ value.