Dulbecco s modified eagle s medium dmem

Cell culture dishes and multi-well plates were purchased from Becton Dickinson Biosciences (Bedford, MA, USA), Thermo Scientific (Waltham, MA, USA), and TrueLine (Nippon Genetics Co. Ltd.; Tokyo, Japan). Dulbecco’s modified Eagle’s medium (DMEM) and alpha modified Eagle’s medium (αMEM) were purchased from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan) and ICN Biomedicals (Aurora, OH, USA), respectively, and fetal bovine serum (FBS) was from Nichirei Bioscience Inc. (Tokyo, Japan). Angiotensin II was from Peptide Institute Inc. (Osaka, Japan). Losartan potassium salt and PD123319 were purchased from Fujifilm Wako Pure Chemical (Osaka, Japan) and Abcam (Cambridge, UK), respectively. MIA was obtained from Sigma-Aldrich (St. Louis, MO, USA).

Human colorectal adenocarcinoma-derived cell line LoVo, of which some loci were different from those of an original LoVo (ATCC: CCL-29), was kindly provided by Kirin corporation. Human colorectal adenocarcinoma-derived cell line HCT116, HT29, and DLD-1, human non-small cell lung cancer (NSCLC) adenocarcinoma-derived cell line A549, and human prostate cancer-derived cell line LNCaP were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). LoVo, HCT116, HT29, DLD-1, and LNCaP cells were maintained in Roswell Park Memorial Institute (RPMI) medium 1640 (RPMI1640; Nissui Pharmaceutical, Tokyo, Japan) containing 10% fetal bovine serum (FBS; JRH Biosciences), 100 U/mL of penicillin G (Sigma #13752), and 0.1 mg/mL of kanamycin (Sigma #K1377). A549 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Nissui Pharmaceutical) supplemented with 10% FBS, 100 U/mL of penicillin G, and 0.1 mg/mL of kanamycin. All cell lines were maintained at 37°C in a 5% CO₂–95% air atmosphere. Unfortunately, we conducted a study using Mycoplasma-infected cells in some experiments.

The contraction of isolated HSCs was measured on a solid gel mixture containing 2.0-μm fluorescent latex beads (Sigma Aldrich, #L4530-1ML). The gel mixture was made with rat tail type I collagen (Millipore, Darmstadt, Germany) and 2.65× Dulbecco’s modified Eagles medium (DMEM, Nissui Pharmaceutical, Tokyo, Japan) and adjusted to pH 7 with 0.5 N NaOH. 150 μL of the gel mixture was plated on a 35 mm glass bottom dish (glass diameter, 14 mm; Matsunami Glass, Osaka, Japan) and flattened. The fluorescent latex beads were diluted 4000-fold with MilliQ water and sonicated for 1 min, and 400 μL of the solution was placed on the gel. The final concentration of collagen in the dish was 1 mg/mL. Collagen was then solidified in a 5% CO₂ incubator at 37°C. Isolated HSCs, which were cultured in DMEM supplemented with 10% fetal bovine serum (Biowest, Cat No: S1820-500, Lot No: S08048S1820), 100 units/mL penicillin, and 100 mg/mL streptomycin and incubated at 37°C in a humidified atmosphere with 5% CO₂ for less than 24 h, were seeded on the solid gel mixture 4 h after the gel preparation. For contraction measurement, the dish solution was replaced by HEPES-buffered solution (125 mM NaCl, 5 mM KCl, 1.3 mM MgSO₄·7H₂O, 1.2 mM CaCl₂·2H₂O, 20 mM HEPES, 5.8 mM D-glucose, pH 7.2–7.4).