Reagents
Talabostat mesylate was purchased from MedChemExpress (Cat. No.: HY-13233A, CAS: 150080-09-4, USA). FAP (Cat. No.: 27596-1-AP), cytochrome c (Cat. No.: 10993-1-AP) and cleaved caspase 3 (Cat. No.: 25128-1-AP) polyclonal antibodies were purchased from Proteintech China. Corning™ transwell™ multiple well plates (12 well plate and pore size of 3 μm, Cat. No.: 07-200-157,) and Lipofectamine 3000 transfection reagent (Cat. No.: L3000008) were purchased from Thermo Fisher Scientific USA. Matrigel was purchased from Corning (Cat. No.: 354234, USA). The reactive oxygen species (ROS) detection kit (Cat. No.: S0033S), lipid oxidation (MDA) detection kit (Cat. No.: S0131S), reduced glutathione (GSH) detection kit (Cat. No.: S0053), annexin V-FITC apoptosis kit (Cat. No.: C1062L), one-step TUNEL apoptosis kit (Cat. No.: C1086) and cell counting kit-8 kit (CCK-8, Cat. No.: C0038) were purchased from Beyotime China. Human FAP Elisa kit was purchased from Abcam China (Cat. No.: ab256404). Organoids were obtained from Shannxi Weiyuan Biomedical Research Institute Co., Ltd. The use processes of human tissues and serum were in accordance with protocols approved by the ethical review committee of Xi’an Jiaotong University (Approval No. LLSBPJ-2023-006) and were obtained through patient's consent. CellTiter-Glo® 3D cell viability assay was purchased from Promega (Cat. No.: G9683, USA). The FAP siRNA (sequence: CCUGAUCGGCAAUUUGUAUTTAUACAAAUUGCCGAUCAGGTT) was purchased from GenePharma China. All other reagents and chemicals were local.
CCK-8 assay
MDA-MB-231 cells (1 × 105 cells/mL each well) were cultured in 96-well plates. For Talabostat groups, Talabostat was added into each well (finally concentrations: 1.0 μM, 5.0 μM, 10.0 μM, 25.0 μM, 50.0 μM, 0.5 % DMSO as co-solvent, the same below) and cells were cultured at 37 °C for suitable time. Then the culture mediums were discarded, and the cell viabilities were obtained according to the standard CCK-8 assay protocol using the BioTek Synergy LX microplate reader, USA. The absorbance of cells treated with 0.5 % DMSO was defined as 100 %. Protocols of cell viabilities of empty vector (EV) and siRNA groups were the same without addition of Talabostat. Unless otherwise stated, all experiments were repeated three times.
Transwell assay
MDA-MB-231 cells (2 × 105 cells/mL each well) were cultured in Corning™ transwell™ multiple well plates (covered by matrigel). For Talabostat groups, Talabostat was added into each well (finally concentration: 25.0 μM) and cells were cultured at 37 °C for suitable time. Then the culture mediums were discarded, cells were washed with clean PBS. And cells on bottom were stained with 0.1% crystal violet. The stained cells were counted and photographed by microscope. The protocols of transwell assays for EV and siRNA groups were the same without addition of Talabostat.
ROS staining
MDA-MB-231 cells (1 × 105 cells/mL each well) were cultured in 96-well plates. Talabostat was added into each well (finally concentration: 25.0 μM) and cells were cultured at 37 °C for 24 hours. Then the culture mediums were discarded, cells were washed with clean PBS. Cells and cell nucleus were stained with DCFH-DA (finally concentration: 50 μM) and PI (finally concentration: 50 μM), respectively at 37 °C. Then cells were imaged by fluorescent microscope.
MDA and GSH levels detections
MDA-MB-231 cells (2 × 105 cells/mL each well) were cultured in 96-well plates. Talabostat was added into each well (finally concentrations: 5.0 μM, 10.0 μM, 25.0 μM) and cells were cultured at 37 °C for 24 hours. Then the culture mediums were discarded, cells were washed with clean PBS. Then cells were lysed and centrifuged, supernatants were collected for MDA and GSH levels detections.The assays were performedaccording the standard protocols of MDA and GSH detection kits and the concentrations of MDA and GSH were determined by BioTek Synergy LX microplate reader, USA.
Transmission electron microscope (TEM)
MDA-MB-231 cells (2 × 105 cells/mL each well) were cultured in 96-well plates, Talabostat was added into each well (finally concentration: 25.0 μM) and cells were cultured at 37 °C for 24 hours. Then the culture mediums were discarded, cells were washed with clean PBS and performed to TEM tests. Results were obtained from Servicebio Technology Co., Ltd. China.
Flow cytometry assays
MDA-MB-231 cells (1 × 108 cells/mL each well) were cultured in 48-well plates, Talabostat was added into each well (finally concentration: 25.0 μM) and cells were cultured at 37 °C for 24 hours. Then the culture mediums were discarded, cells were washed with clean PBS and performed to Flow Cytometry tests. The experiments were performed according to the standard protocols of annexin V-FITC apoptosis kit through NovoCyte Flow Cytometry (Agilent, USA).
TUNEL staining
The TUNEL staining results were obtained according to one-step TUNEL apoptosis kit protocols through Leica TCS SP5 laser scanning confocal microscope (Leica, Germany).
Western blot
All samples were prepared according to the optimized protocols. Some key conditions: the loading qualities of proteins were about 10 μg and the loading volumes were completed by 1×Loading Dye. The antibodies were diluted about 5000 times for experiments. The current of transfer film was about 250mA.
Whole genome RNA expression sequencing
The whole genome RNA expression sequencing (RNA-seq) results were obtained from Majorbio, China.
Enzyme-linked immunosorbent assay of FAP
The human serums were diluted 50 times for subsequent Elisa tests. The procedures were strictly followed by the kit protocols.
Cell viabilities of organoids
The cell viabilities of organoids were obtained according to the protocols of CellTiter-Glo® 3D cell viability assay. Organoids were treated with Talabostat (finally concentration: 50 μM) at 37 °C for 24 hours. Then the culture mediums were discarded, organoids were washed with clean PBS and performed for cell viability assays. The luminescence of organoids treated with 0.5 % DMSO was defined as 100 %.
siRNA transfection
MDA-MB-231 cells (1 × 108 cells/mL each well) were cultured in 24-well plates. siRNA and Lipofectamine 3000 transfection reagent were mixed gently for use (the finally concentration of siRNA is 200 nM). The mixed solutions were then added in each well, and the cells were incubated at 37 °C for 48 hours. After that, the mediums were discarded and cells were washed with clean PBS and replaced with fresh mediums for further incubation. Western blot assays were used to verify the protein knock down efficiency.