Caco 2

Human colon adenocarcinoma (Caco-2) cells obtained from ATCC (Manassas, VA, USA) were cultured in Dulbecco’s Modified Eagle Medium (DMEM), high-glucose supplemented with 20% FBS, 4 mM L-glutamine, and 20 mM non-essential amino acids, 100 U/mL penicillin, and 100 μg/mL streptomycin. Madin–Darby canine kidney (MDCK) cells obtained from AddexBio Technologies (San Diego, CA, USA) were maintained in minimum essential medium (MEM) supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. To generate MDCK cells overexpressing hOAT1 (hOAT1-MDCK) and hOAT3 (OAT3-MDCK), the parent MDCK cells were transfected with the expression plasmids of hOAT1 (# SC303532) or hOAT3 (# SC122673) or an empty pCMV6 vector (OriGene Technologies, Inc., Rockville, MD, USA). After transfection, the hOAT1-MDCK and hOAT3-MDCK cells were selected by complete MEM containing 700 µg/mL G418 for eight weeks. The Chinese hamster ovary (CHO-K1) cells obtained from CLS (Deutschland, Germany) were cultured in complete Kaighn’s modification of Ham’s F-12 (with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin). The CHO-K1 cells overexpressing hOCT1, hOCT2, hMATE1, and hMATE2 were generated by the transfection of parent CHO-K1 cells with the expression plasmids of hOCT1 (#RC211872), hOCT2 (#RC207921), hMATE1 (#RC201890), and hMATE2 (#RC223318) or an empty pCMV6 vector (OriGene Technologies, Inc., Rockville, MD, USA). Transporter-overexpressing cells were selected with 3 mg/mL of G418. Cells were cultured at 37 °C in a humidified atmosphere containing 5% CO₂.

We purchased mouse transgenic sertoli cell line 15P-1, IECs, MC38 (Murine adenocarcinoma), and Caco2 (Human colorectal adenocarcinoma) from ATCC, which uses PCR-based assays, karyotyping, and other methods to confirm the identity of cells. MC38 and 15P-1 were cultured in Dulbecco’s modified Eagle’s medium, whereas Caco2 cells were grown in an EMEM containing 10% FBS (Thermo Fisher Scientific) in a humidified atmosphere with 5% CO₂ at 37°C.

HCT116 (#CCL-247, RRID: CVCL_0291), SW480 (#CCL-228, RRID: CVCL_0546), SW620 (#CCL-227, RRID: CVCL_0547), and Caco-2 (#HTB-37, RRID: CVCL_0025) were obtained from ATCC. LOVO (RCB1639, RRID: CVCL_0399) and COLO320 (RCB1193, RRID: CVCL_1989) were obtained from RIKEN BRC. Cells were maintained in high-glucose DMEM (Gibco) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin (Lonza), and 10% FBS (HyClone). Cells were maintained in the culture for maximum of 15 passages. Cells were monitored monthly for Mycoplasma contamination, and cell lines were authenticated using short tandem repeat analysis (Labcorp).

The human colorectal adenocarcinoma cell line Caco‐2 was obtained from ATCC and cultured in modified Eagle medium (MEM) containing 20% fetal bovine serum, 1% non‐essential amino acid, and 1% penicillin‐streptomycin (Gibco, Thermo Fisher Scientific). The cells were incubated at 37°C in a humidified atmosphere of 5% CO₂. When reaching 80% confluence, the cells were treated with various concentrations of 5‐HL (0, 0.2, and 0.7 µmol/L), PG (0, 15, and 50 µmol/L), or 3‐MH (0, 5, 10, and 20 µmol/L) for 12 h prior to analysis.
More experimental details were provided in Supporting Information.

The human colon adenocarcinoma cell line Caco-2 and human pancreatic carcinoma cell line PANC-1 were purchased from the American Type Culture Collection (ATCC, Virginia, USA). The Caco-2 and PANC-1 cells were cultured in Dulbecco’s modified Eagle’s medium with 10% fetal calf serum and placed in a 37 ℃, 5% CO₂ incubator (Thermo Scientific).
A Caco-2 and PANC-1 co-culture system was established using a 0.4 μm polyester transwell chamber and a matching 6-well plate. The PANC-1 cells with 10 nM CAE and 0.5 mM palmitic acid (PA) (Sigma-Aldrich, Missouri, USA) were incubated in the co-culture system with Caco-2 cells. The control group was incubated with equal amounts of saline and fatty acid-free bovine serum albumin.

The human colorectal adenocarcinoma (Caco-2), human colorectal adenocarcinoma (HT29), normal human intestinal (I407), human cervical cancer (HeLa), human breast cancer (MCF7), human bone osteosarcoma (U2OS), and human acute T-cell leukemia (Jurkat J6) cell lines were purchased from American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were cultured in RPMI 1640 medium (Labtek Eurobio, Milan, Italy), supplemented with 10% FCS (Euroclone, Milano, Italy) and 2 mM L-glutamine (Sigma-Aldrich, Milano, Italy), at 37 °C and in a 5% CO₂ atmosphere. The compounds were dissolved in DMSO in a 30–40 mM stock solution. In cell treatments, the final DMSO concentration never exceeded 0.1%.