5 fluorouracil 5 fu

10-HDA (Herbourify Co., Ltd., Chengdu, China) and 5-fluorouracil (5-FU; MedChemExpress, Princeton, NJ, USA) were dissolved in 100% dimethyl sulfoxide (Sigma-Aldrich, St. Louis, MO, USA) to obtain a 20 mM stock solution, and stored at -20°C before use. 2′,7′-Dichlorofluorescein diacetate (DCFH-DA; Merck Chemicals Shanghai Co., Ltd., Shanghai, China), an Apoptosis and Necrosis Assay Kit, an Annexin V-FITC Apoptosis Detection Kit, a Mitochondrial Membrane Potential Assay Kit with JC-1, and N-acetyl-L-cysteine (NAC) were purchased from Beyotime Institute of Biotechnology (Shanghai, China). The DNA Content Quantitation Assay (Cell Cycle) and Cell Counting Kit-8 (CCK-8) were purchased from Solarbio (Beijing, China). All of the antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). Other chemicals were of analytical grade.

Cell Counting Kit-8 (CCK-8) was used to evaluate MPSSS-induced cytotoxicity. The SVEC4-10 cell line was used as lymphatic endothelial cells in this study. CT-26 CAFs and SVEC4-10 cells were seeded into 96-well plates at a density of 3,000 cells/well, and incubated with MPSSS at different concentrations for 24 h. 5-Fluorouracil (5-FU) (MedChemExpress, USA) of different concentrations (0-10 µg/ml) were also used to treat CAFs as positive control. Then, 10 µL of the CCK-8 reagent was added to each well and incubated for 2 h. The absorption at 450 nm was measured with a microplate reader. To detect the effect of MPSSS-treated CAF supernatants on the proliferation of SVEC4-10 cells, SVEC4-10 cells were cultured in 96-well plates at a density of 3,000 cells/well, and treated with media contained 30% CAF supernatants. CAFs were seeded in 6-well plates at a density of 5 × 10⁵ cells/well. Different treatments were added after the cells adhered to the plates. After 24 h of stimulation, supernatant was collected and centrifuged. CAFs treated with 30 µg/ml MPSSS or inhibitors were used as the experimental group, and PBS was used as the negative control.

5-Fluorouracil (5-Fu) was obtained from MedChemExpress. Recombinant TIMP-2 was obtained from PeproTech (diluted to 10 ng/mL in the experiment). The antibody for TIMP-2 neutralization was obtained from R&D systems (diluted to 5 μg/mL in the experiment). Antibodies to MAPK (Erk1/2) (Cat No.4695), phospho-MAPK (Erk1/2) (Thr202/Tyr204) (Cat No.4370), Erk5 (Cat No.3552), phospho-Erk5 (Thr218/Tyr220) (Cat No.3371) and GAPDH (Cat No. 97166) were purchased from Cell Signaling Technology (CST). In the WB experiment, the above antibodies were diluted 1:1000. HRP-conjugated antibodies were obtained from Hangzhou Fude Biological Technology.

Cell-level dimethyl sulfoxide (DMSO) (Solarbio, Beijing, China) was used to dissolve JAC (HerbPurify, Chengdu, China) and 5-fluorouracil (5-FU) (Med Chem Express, Princeton, NJ, USA) and the solutions were stored at −20°C. Cell Counting kit-8 (CCK-8), Annexin V-FITC/PI apoptosis kit, MMP assay kit, and DNA quantification kit were acquired from Solarbio. ROS assay kit was obtained from Beyotime (Shanghai, China).

Dimethyl sulfoxide (DMSO), FAO activator bezafibrate (BZF) and inhibitor etomoxir (ETO), glycolysis inhibitor 2-Deoxy-D-glucose (2-DG), glutaminase inhibitor BPTES, P300 inhibitor C646 and 5-fluorouracil (5-FU) were all purchased from MedChemExpress, New Jersey, USA.

HL60, THP-1, MV4-11, HEL, K562, and 293 T cells were obtained from Shanghai Institute of Cell Science, Chinese Academy of Sciences. All cell lines were cultured in RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS and were maintained at 37 °C in 5% CO₂. All cells were subjected to short tandem repeat profiling and an incubation period of no more than two months. Ara-C, adriamycin (ADM), DDP, paclitaxel, 5-fluorouracil (5-FU), and methotrexate (MTX) were purchased from MedChemexpress CO., Ltd. Ara-C-resistant HEL (HLE-R) and HL60 (HL60-R) cells were generated by culturing cells in medium with a gradient of progressively increasing drug concentrations, with a final concentration of 8 μM. For all assays, drug-resistant cells were cultured for 48 h in drug-free medium before use in experiments.