Caco 2

The human colon cancer cell line Caco-2 (RIKEN Cell Bank, Tsukuba, Japan) spontaneously differentiates into an enterocyte-like phenotype and serves as a model for intestinal epithelial cells [29] . We cultured Caco-2 cells in low-glucose DMEM supplemented with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin at 37 °C under a 7% CO₂ atmosphere for 28 days as we previously described [30] (link). The medium was changed every 2 days, and the cells were incubated in serum-free DMEM for 24 h before all experiments. Dimethyl sulfoxide (DMSO) was the control vehicle, and the solvent (final concentration 0.1% v/v) for microcystin-LR, SP600125, rifampicin, and probenecid.

Human cervical carcinoma HeLa (RCB0007), hepatoma HepG2 (RCB1648), colon carcinoma Caco-2 (RCB0988), embryonic kidney 293T (RCB2202), and mouse myoblast C2C12 (RCB0987) cells were obtained from Riken Cell Bank (Tsukuba, Japan). Human embryonic kidney 293 (JCRB9068), rat hepatoma Fao (89042701), and rat cardiomyocyte H9c2 (CRL-1446) cells were obtained from Japanese Collection of Research Bioresources (JCRB) Cell Bank (National Institutes of Biomedical Innovation, Health and Nutrition, Osaka, Japan), ECACC (Salisbury, UK), and ATCC (Manassas, VA, USA), respectively. These cells were maintained in Eagle’s minimum essential medium (MEM, FUJIFILM Wako Pure Chemical Corporation) containing antibiotics and 10% fetal bovine serum, except for C2C12 and H9c2 cells, which were maintained in Dulbecco’s modified Eagle medium (FUJIFILM Wako Pure Chemical Corporation). Primary cultured rat cardiomyocytes (CMC02) were obtained from COSMO BIO (Tokyo, Japan).

The human colonic adenocarcinoma epithelial cell line Caco-2 was obtained from Riken Cell Bank (Ibaraki, Japan) and kept in a humidified incubator at 37°C with 5% CO₂. Caco-2 cells were maintained in Dulbecco's Modified Eagle Medium (DMEM)-High glucose (Wako, Osaka, Japan) supplemented with 10% heat-inactivated (30 min, 56°C) fetal bovine serum (Biowest, Nuaillé, France), 100 U/mL penicillin, 100 μg/mL streptomycin, and 1% nonessential amino acids (NEAA) (Wako). In this study, Caco-2 cells were used between passages 25 and 40. Cells cultivated to 80% confluence were seeded on 12-well Transwell® inserts (1.12 cm² polycarbonate membrane with 0.4 μm pore size; Corning Life Sciences, Corning, NY) at a density of 5 × 10⁴ cells per well. The culture medium was changed every 2 days until 20 days later when full polarization of the Caco-2 cell monolayer was achieved. The integrity of the Caco-2 cell monolayer was evaluated by measuring the transepithelial electrical resistance (TER) using a milicell-ERS (Millipore, Billerica, MA). The TER values measured in the experiments were summarized as table form below each figure, and all the figures were expressed as the relative TER value compared to the value before experimental treatment.