OP-B (CAS: 38971-41-4) and OP-D (CAS: 41753-55-3) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd (Shanghai, China). OP-B and OP-D were dissolved in DMSO as a 10 mmol/l stock solution and stored at 4 ℃. Inhibitor INF39 and Belnacasan (CAS: 866028-26-4, 273404-37-8) were purchased from Med Chem Express. Polo-188 (CAS:9003-11-6) was purchased from Beijing solarbio science﹠technology co., Ltd.
A549 and NCI-H460 cells were obtained from the Stem Cell Bank, Chinese Academy of Sciences (Shanghai, China). A549/DDP and A549/PTX cells were kind gifts from Professor Zhigang Guo (NanJing Normal University). A549, NCI-H460 cells were cultured in DMEM/F12 medium (Gibco, Australia), and A549/DDP, A549/PTX cells were cultured in RPMI 1640 medium (Gibco, Australia) with 10% fetal bovine serum (FBS; Gibco, Australia), supplemented with 1% penicillin/streptomycin solution (Gibco, Australia). All of the cells were maintained at 37 °C in humidified atmosphere of 5% CO2.
Cell viability assays
The inhibition of OP-B on A549, NCI-H460, A549/PTX, A549/DDP cells was estimated by Cell Counting Kit-8 (CCK-8; Dojindo, Beijing, China) as described previously (16).
Western blot analysis
The cells were lysed in radioimmunoprecipitation assay (RIPA) buffer (Beyotime, Shanghai, China) containing 1% phenylmethanesulfonyl fluoride (PMSF) before suspended in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) sample loading buffer (Beyotime, Shanghai, China), then separated on 12% SDS-PAGE (Beyotime, Shanghai, China) and transferred onto polyvinylidene fluoride (PVDF) membranes (Thermo Fisher, US). After the membranes were blocked with 5% non-fat milk, they were incubated at 4 °C overnight with primary antibodies against Caspase-1 (1:500, CST, US), GSDMD (1:500, CST, US), GSDMD-N (1:500, CST, US), NLRP3 (1:1000, CST, US), and β-actin (1:2000, CST, US). After incubation with horseradish peroxidase (HRP)-linked anti-rabbit (1:2000, CST, US) or anti-mouse (1:2000, CST, US) secondary antibodies for 2 h at room temperature, the bands were detected with a Gel Doc™ XR+ Gel Documentation System (Bio-Rad, US) with enhanced chemiluminescence (ECL) reagents (Bio-Rad, US).
Quantitative real-time PCR (qRT-PCR)
Total RNA was extracted from A549 or A549/DDP cells using TRIzol reagent (Sangon Biotech) according to the manufacturer’s protocol. Then, the RNA was reverse transcribed to cDNA using PrimeScript™ RT reagent Kit with gDNA Eraser (Takara). Quantitative real-time PCR was performed using cDNA primers specific for mRNA. The gene GAPDH was used as an internal control. All the real-time PCR reactions were performed using Takara′s SYBR Premix Ex Taq™ II (Tli RNaseH Plus) in Applied Biosystems 7500 Fast Real-Time PCR System (Applied Biosystems). The 2-△△Ct method was used for quantification and fold change for target genes was normalized by internal control.
Cells at a density of 1×104 cells/well were seeded into 96-well plates. After exposure to the indicated treatments, the cells were fixed with 4% paraformaldehyde for 15 min at room temperature, then blocked with 5% goat serum and 0.3% Triton X-100 in phosphate-buffered saline (PBS) for 1 h. After that, the cells were incubated with a primary antibody against Cox-2 (1:200, CST, US), IL-1β (1:200, CST, US) in antibody dilution buffer (ADB; 1X PBS/1% bovine serum albumin (BSA)/0.3% Triton X-100) overnight at 4 ℃. Then, the cells were incubated with a fluorochrome-conjugated anti-rabbit secondary antibody (1:1000, CST, US) in ADB for 2 h at room temperature in the dark. Subsequently, the cells were stained with DAPI (1 μg/ml, CST, US) for 5 min. Images were obtained under a fluorescence microscope (TCS SP8, Leica).
When frozen sections were used for immunofluorescence, the sections were first blocked with goat serum; the rest of the procedure was the same as that for the goat serum-blocked cells.
LDH Release Assay
The activity of LDH released into cell culture supernatants was detected using the CytoTox 96 Non-Radioactive Cytotoxicity Assay Kit (Omega) according to the manufacturer’s protocol for analyzing pyroptosis.
In vivo xenograft assay (nude mice models and xenograft zebrafish)
The BALB/c nude mice (4-week old) were maintained under specific pathogen‑free (SPF) conditions. Animal welfare and experimental procedures were performed in compliance with the National Institutes of Health Guidelines for the care and use of laboratory animals, and all protocols were approved by the Ethics Review Committee of Nanjing University of Chinese Medicine. To establish the orthotopic xenograft lung cancer model, the luciferase-expressing A549 or A549/DDP cell line with lentivirus was established, then A549 or A549/DDP cells (2×107 in 0.2ml medium of a 1:1 mixture of RPMI 1640 and Matrigel 354,248) were injected into right lung parenchyma of the mice, and the volume of tumors were monitored by luciferase imaging of live animals using an IVIS Spectrum bioluminescence imaging system (PerkinElmer, US) after intraperitoneal injection of 200 μl D-Luciferin substrate (15 mg/ml in DPBS, PerkinElmer). And the mice were mainly used to test the toxicity and pharmacological activity of OP-B on A549 or A549/DDP xenograft mice. The mice for toxicity-detection were divided into 5 groups (6 in each group), including polo-188 group (62.5mg/ml Poloxamer), OP-B groups (1.5 or 3mg/Kg OP-B), and the Normal or Mock group (saline). And the mice for pharmacological activity detection were divided into 9 groups, including the Normal group (saline), A549 or A549/DDP Mock groups (saline), A549 OP-B-treatment groups (1.5 or 3mg/Kg OP-B), A549/DDP OP-B-treatment groups (1.5 or 3mg/Kg OP-B), and A549 or A549/DDP cyclophosphamide groups (20mg/Kg cyclophosphamide). All of the mice were treated with intraperitoneal injection (i.p. daily, n=28). The polo-188 was formulated with 0.9% NaCl, and OP-B was formulated with polo-188. 25 days later, all mice’ hearts, livers, lungs and kidneys were harvested, then half of the tissues were used for GPT, GOT and CRE detectinon with microplate test kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), and the rest of them were used for haematoxylin-eosin (H&E) staining, Transmission Electron Microscope (TEM) observation, or immunohistochemistry and immunofluorescence observation.
AB/wt zebrafish embryos were raised at 28 °C in fish water. At 48 hours fertilization (hpf), A549 cells, labeled with a red fluorescent dye for cell viability (Cell Tracker™ CM-DiI, Invitrogen, CA, USA) and resuspended in HBSS were injected into the yolk sac of zebrafish embryos (200 cells/embryo). Then, embryos were incubated at 34 °C. At 72 h post injection, the proliferation of tumor cells was evaluated through a fluorescence stereomicroscope (OLYMPUS U-HGLGPSD, equipped with Cell Sens Entry software, Tokyo, Japan). The software Image J was used to quantify the proliferation rate of tumor cells. The protocols for the animal experiments were approved by the Ethics Review Committee of Nanjing University of Chinese Medicine.
Transmission Electron Microscope (TEM) assay
The model and OP-B groups were selected for TEM analysis to observe the morphology of A549 cells and A549/DDP cells. The mice were sacrificed, 1/3 of tissue was removed from lung and fixed with 3% glutaraldehyde for more than 2 hours, then immobilized with 1% osmium acid for 2 hours. The tissues were dehydrated step by step with ethanol and acetone and then soaked overnight and embedding agent. After the tissues were embedded, polymerized, repaired, sliced and double stained with uranium acetate and lead citrate, they were observed by TEM（JEM-1011）.
Data entry and all analyses were performed in a blinded fashion. All statistical analyses were performed using GraphPad Prism7.0 software. Statistical significance was calculated using two-tailed unpaired t-test on two experimental conditions or two-way ANOVA when repeated measures were compared, with p < 0.05 considered statistically significant. All graphs show mean values ± SEM.