Hct116 colon cancer cells

HCT116 colon cancer cells (American Type Culture Collection, Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (including 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin) at 37 °C in a 5% CO₂ atmosphere.

Mouse melanoma B16F1 and B16F10 cells and human HCT116 colon cancer cells were obtained from American Type Culture Collection (Manassas, VA) and cultured in RPMI medium supplemented with 10% FBS, 1 mM sodium pyruvate, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, and 100 U/ml penicillin-streptomycin in a humidified atmosphere of 5% CO₂ at 37°C. Human umbilical vein endothelial cells (HUVECs) were maintained and cultured in M199 as described previously [19 (link)], and only passages 2-7 were used for all experiments. Human multiple myeloma IM-9 and RPMI 8226 cells were obtained from Korean Cell Line Bank (Seoul, Korea). The cells were cultured in RPMI-1640 medium in a humidified CO₂ incubator. Cell viability or cytotoxicity was evaluated by 3-[4,5-cimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT, Sigma-Aldrich) assay.

Human MDA-MB-231 breast cancer cells and HCT116 colon cancer cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (FBS, Invitrogen, Eugene, OR). Cells were cultured at 37°C with 5% CO₂ in a humidified incubator.

Hepa 1-6 hepatocellular carcinoma cells, HCT-116 colon cancer cells and HEK 293T cells were obtained from the American Type Culture Collection (ATCC), and primary mouse embryonic fibroblasts (MEFs) were purified from mouse embryos at E14.5. A green fluorescent protein (GFP)-encoding vector was transfected into HCT-116 cells and stable expression of GFP was established. All cells were maintained in DMEM high glucose medium with 10% FBS and 100 U/mL penicillin and streptomycin at 37 °C under 5% CO₂.

HCT-116 colon cancer cells, MCF-7 and MDA-MB-231 breast cancer cells, UM-UC-3 bladder cancer cells, and HeLa cervical cancer cells were obtained from the American Type Culture Collection (ATCC^®, Manassas, VA, USA). All batches of culture media were supplemented with 10% (v/v) of fetal bovine serum (Life Technologies, Carlsbad, CA, USA), 100 U/mL penicillin, 100 μg/mL streptomycin and 0.25 μg/mL amphotericin B (Sigma). HCT-116 colon cancer cells, MCF-7 and MDA-MB-231 breast cancer cells were cultured in Dulbecco´s Modified Eagle´s Medium (DMEM, Sigma). UM-UC-3 bladder cancer cells were cultured in Eagle´s Minimum Essential Medium (EMEM; Corning, NY, USA) with 1.5 g/L sodium bicarbonate, non-essential amino acids, L-glutamine and sodium pyruvate. HeLa cervical cancer cells were cultured in DMEM (Corning) with 4.5 g/L glucose, and L-glutamine without sodium pyruvate. All cells were maintained at 37°C in a 5% CO₂ humidified atmosphere.

Doxorubicin hydrochloride (DOX; Product Number: D1515; CAS Number: 25316-40-9; formula: C₂₇H₂₉NO₁₁.HCL) was obtained from Sigma–Aldrich Chemicals (St. Louis, MO, USA). DOX has a molecular weight of 579.98 g/mol and is in the form of powder. Methanol (CH₃OH) and chloroform (CHCl₃) were purchased from Sigma–Aldrich. Dulbecco’s Modified Eagle’s medium (DMEM; Lot No: 09221712, GenDEPOT, St. Louis, MI, USA) and 10% (v/v) fetal bovine serum (FBS; Lot No: WB0009) were purchased from GE Healthcare Life Science (Queensland, Australia). HCT116 colon cancer cells were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA). TSP-d₄ is a 3-(trimethylsilyl) propionic acid that was purchased from Cambridge Isotope Laboratories in the United States.

We purchased mycoplasma-free HCT116 colon cancer cells (American Type Culture Collection, Manassas, VA, USA). The supplier authenticated the cells through short tandem repeat analysis (Cell Line Authentication Service, American Type Culture Collection, Manassas, VA, USA) and Sanger sequencing. HCT116 cells are male. We extracted DNA from these cells directly from the supplier without culturing.