Dulbecco modified eagle medium (dmem)

DMEM, trypsin-EDTA solution, paraformaldehyde, Triton-X 100, Hoechst33258 and 2,5-diphenyltetrazolium bromide (MTT) were purchased from Beyotime Biotechnology (Beijing, China). Cell cycle detection kit and BrdU cell proliferation detection kit was purchased from KeyGEN BioTECH. Fetal bovine serum was purchased from Gbico (USA). Lipfectamine 2000 Reagent was obtained from Invitrogen (Thermo Fisher Scientific, US); Reverse Transcription Kit and SYBR Premix ExTaq^TMII Kit were purchased from TaKaRa (Dalian, China). SanPrep EndoFree Plasmid Kits, antibody of CDK1, CDK2, CDC25A, CDC25C, CCNB1, PLK1, Wee1, p-CDK1 (phospho Tyr15) and GAPDH and PCR primers were obtained from Shangon Biotech (Shanghai, China). Monoclonal mouse antibody p-CCNB1 (phospho Ser147), p-CDC25C (phospho Ser216), anti-rabbit IgG-conjugated Alexa 594 antibody and anti-His-tag antibody were obtained from Proteintech Company (Wuhan, China).

The present study employed human bladder cancer cells (5637, UMUC-3, and T24), along with normal human uroepithelial cells (SVHUC). Roswell Park Memorial Institute-1640 medium (RPMI-1640; Procell Life Science & Technology, Wuhan, China) was used to culture 5637 and T24 cells, and Dulbecco’s Modified Eagle’s Medium (DMEM; Procell Life Science & Technology, City, US State abbrev. if applicable, Country) was used to culture UMUC-3 and SVHUC cells. The China Center for Type Culture Collection (CCTCC, No: GDC078; Wuhan, China) provided all of the cells. The cell cultures were grown within a cell incubator including 5% CO₂ under the condition of 37 °C using RPMI-1640 medium and DMEM that contained 1% penicillin-streptomycin (ST551; Beyotime, Wuhan, China), as well as 10% fetal bovine serum (FBS; Ausbian Corporation, Australia).

H9c2 cardiomyoblasts were cultured in DMEM containing 10% (v/v) FBS and 1% penicillin–streptomycin at 37 °C in a moist incubator with 5% CO₂. The cells were subcultured when cell density was about 80–90% and divided into control group, DOX group, DXXK 800 ng/mL group, DXXK 400 ng/mL group and DXXK 200 ng/mL group. The DOX group was given DOX 4 μ mol/mL for 24H; the DXXK groups were given DOX 4 μ mol/mL + DXXK 800 ng/mL, DOX 4 μ mol/mL + DXXK 400 ng/mL and DOX 4 μ mol/mL + DXXK 200 ng/mL, respectively. H9c2 rat cardiomyocytes were purchased from GIBCO; THERMO FISHER SCIENTIFIC, INC.; FBS (Beyotime, Shanghai, China), penicillin–streptomycin (Beyotime, Shanghai, China) and DMEM (Beyotime, Shanghai, China).

To test the migration ability of breast cancer cells, suspensions of MCF7 and MDA-MB-231 cells were prepared in DMEM without FBS at a density of 2 × 10⁴ cells/mL. The upper chamber of the Transwell was filled with 100 μL of this suspension, while the lower chamber was filled with 500 μL of DMEM containing FBS. The excess cells were retrieved with a sterile cotton swab, fixed in 4% formaldehyde, and stained with 0.1% crystal violet to count the number of migrating cells.
To investigate the invasiveness of MCF7 and MDA-MB-231 cells, the stroma was diluted in DMEM without FBS at a ratio of 1:7 (Beyotime, Guangzhou, China). An aliquot of 50 μL was aspirated into the upper chamber and allowed to rest for 3–4 h. The rest of the procedure was the same as that described for the migration assay.

GL-V9 (C₂₄H₂₇NO₅,(5-hydroxy-8-methoxy-2-phenyl-7-(4-(pyrrolidin-1-yl) butoxy)4H-chromen-4-one), MW 409.47, purity 99%) is a new flavonoid derivative and synthesize by Prof. Zhiyu Li in our lab [25] (link). GL-V9 was dissolved in dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA), made into the parent solution with the concentration of 0.1 M, and then stored at − 80 °C. The final concentration is 2.5, 5, 10, 15 and 20 μM, diluted in Dulbecco's modified Eagle medium (DMEM; GIBCO, Carlsbad, CA, USA).
Paclitaxel injection was purchased from Haikou Pharmaceutical Factory. Dorsomorphin Dihydrochloride (Compound C, C₂₄H₂₇ClN₅O, MW 472.41 and purity 99.73%) was purchased from MedChem Express and dissolved in DMSO to 5 mM. N-acetyl-L-cysteine, which was from Beyotime Biotechnology, was dissolved in water to 0.5 M and was diluted with DMEM to its final concentration. Etomoxir (C₁₅H₁₈ClNaO_4, MW 320.74 and purity 98.63%) was from MedChem Express and dissolved in water to 10 mM. Dehydroepiandrosterone (DHEA, C₁₉H₂₈O₂, MW 288.42) was from Solarbio Life Sciences and dissolved in DMSO to 1 M.

Human ESCC lines Eca-109 and TE-10 (Chinese Academy of Sciences in Shanghai, Shanghai, China) were highly differentiated squamous cell carcinoma lines. The cells were cultured in DMEM (Beyotime, Shanghai, China) containing 100 mg/ml streptomycin/penicillin, 10% fetal bovine serum (FBS), and 100 U/ml penicillin in an incubator with 5% CO₂ at 37°C. IL-32 interference and overexpression lentivirus (Genechem Company, Shanghai, China) were purchased for transfection. After stable transfection, the cells were subcultured and prepared for follow-up experiments.