Cell cycle and apoptosis detection kit

Cell cycle and cell death were measured using Cell Cycle and Apoptosis Detection Kit (Beyotime; catalog no.: 1052), according to the manufacturer’s instructions. Briefly, transfected 293T cells were grown on a well in a 6-well plate for 36 h. Cells were washed with cold PBS, fixed in 70% ethanol, and stored at 4 °C for subsequent cell cycle analysis. Fixed cells were washed with PBS once and then resuspended in 1 ml of propidium iodide staining reagent. Samples were incubated in the dark for 30 min before cell cycle analysis. The distribution of cells in the cell cycle was measured by flow cytometer (BD FACSCalibur), and quantitation of cell cycle distribution was performed using Multi-cycle Software (ModFit software). The percentage of cells in the G1, S, and G2 phases and cell death was calculated.

Following the instructions, cell cycle and apoptosis detection kit (Beyotime, China) were used for cell cycle and apoptosis measurement. For cell cycle detection, cells were then re-suspended in PI staining solution for 30 min at 37 °C. For apoptosis detection, cells were stained with 5 μM Annexin V-FITC at 4 °C for 15 min, fixed in 70% ethanol overnight at 4 °C and then washed by PBS. The cells were then re-suspended in PI staining solution for 30 min at 37 °C. FACSCalibur flow meter with CellQuest software 3.2 (BD Biosciences) was used for data analysis.

Caspase-3 inhibitor II (Z-DEVD-FMK) was purchased from Calbiochem. Terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) was purchased from Roche. SYBR Green real-time polymerase chain reaction (PCR) master mix was purchased from TaKaRa (Shiga, Japan). The universal type SP kit and DAB kit were purchased from ZSGB-BIO. The Annexin V-FITC apoptosis detection kit and the cell cycle and apoptosis detection kit were purchased from Beyotime Biotechnology. The plasmids (GV230 and GV230-CDH1) were designed and synthesized by Shanghai Genechem Co., Ltd. The monoclonal antibody directed against the E-cadherin cytoplasmic domain (clone 36) was purchased from Transduction Laboratories (Lexington, KY). The polyclonal antibodies directed against H. pylori were purchased from Abcam (ab187301). The polyclonal antibodies directed against pro-caspase-3, caspase-3 (active), Bcl-2, and Bax were purchased from Sangon Biotech. The monoclonal antibody directed against GAPDH was purchased from Beyotime Biotechnology. HRP-anti-mouse antibody and HRP-anti-rabbit antibody were purchased from Boster Biological Technology. Cy3-labeled donkey anti-mouse antibody was purchased from Sangon Biotech.

Cell cycle and apoptosis of cells were detected by BD FACS Canto II. Apoptosis was detected with APC Annexin V Apoptosis Detection Kit (eBioscience) with 7-AAD according to Manufacturer's instructions. Cell cycle of cells was detected with Cell cycle and apoptosis detection kit (Beyotime).

Triptolide was purchased from various commercial sources, including Tansoole and Meilunbio. All general chemicals were purchased from Adamas‐beta or Energy Chemical in the highest purity available and were used without further purification. The cell cycle and apoptosis detection kit, the total cell lysates (C1062L, Beyotime), the BCA protein concentration assay Kit, and the TUNEL cell apoptosis detection kit were purchased from Beyotime Biotechnology. The synthesized compounds were analyzed using a Bruker 400 MHz nuclear magnetic resonance (NMR) spectrometer. High‐resolution mass spectra (HRMS) were recorded using a ZAB‐HS spectrometer with electrospray ionization (ESI). High‐performance liquid chromatography (HPLC) analysis was performed using a Hanbo Sci&Tech NS4201 system equipped with an NU3000 serial UV/Vis detector and two NP7000 serial pumps. The binding affinity was investigated using a Biacore T200 SPR instrument (Cytiva, Sweden). The degrees of apoptosis and cell cycle progression were detected using a NovoCyte3130 flow cytometer (ACEA). Imaging was performed using confocal laser‐scanning microscopy (Nikon A1R).