Nih3t3

The mouse fibroblast-like cell line NIH/3T3 (RCB2767) was obtained from the RIKEN BioResource Research Center (Ibaraki, Japan). Cells were maintained in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12; Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS; HyClone Standard Fetal Bovine Serum Collected and Processed in USA, Cytiva, Tokyo, Japan) and 1% penicillin/streptomycin (Thermo Fisher Scientific) at 37 °C under humidified 5% CO₂ atmosphere.

The mouse embryonic fibroblast cell line NIH 3T3 was purchased from the Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan. A complete medium consisting of Dulbecco’s modified eagle medium (DMEM, Gibco, Waltham, MA, USA) and 10% calf serum (CS, Invitrogen, Carlsbad, CA, USA) was used for cell culture. The cells were incubated in tissue culture polystyrene flasks (Corning, Corning, NY, USA) in 5% CO₂ at 37 °C until a density of 5~6 × 10⁴ cells/cm² was reached. For surface coating, gelatin (Sigma, St. Louis, MO, USA) and poly-l-lysine (Sigma, St. Louis, MO, USA) were diluted in 1× phosphate-buffered saline (PBS) into concentrations of 0.1% (w/w) and 0.1 μg/mL, respectively, as recommended by the manufacturer. Coomassie Brilliant Blue (Bionovas, Toronto, ON, Canada) was diluted into a concentration of 2.5 g/L (in 0.1 L acetic acid, 0.3 L methanol, and 0.6 L ultrapure water) for verifying the presence of gelatin and poly-l-lysine.

Mouse fibroblast cell line NIH3T3 cells were obtained from RIKEN BRC (Tsukuba, Japan) and were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum and Penicillin G / Streptomycin under 5% CO₂ at 37 °C. Cells were incubated with transforming growth factor β (TGFβ) (Millipore Sigma, Burlington, MA, USA) at 10 ng·mL⁻¹ or dimethyl sulfoxide (DMSO) as a vehicle. In some experiments, cells were pretreated with DGK inhibitor R59949 (Millipore Sigma) at 10 μm. Cells were harvested at 0, 30 min, and 6 h of incubation after TGFβ treatment for immunoblot analysis.

Cells lines were obtained from the American Type Culture Collection (ATCC) or Bioresource Collection and Research Center (BCRC, Taiwan), including human breast cancer cell lines MCF-7 (MEM) and MDA-MB453 (DMEM), the human renal carcinoma cell 786-O (RPMI), human embryonic renal epithelial cell 293T (DMEM), human prostate cancer cell lines LNCaP (RPMI), PC3, and PC3-Luc (F-12), human bladder cancer cell T24 (RPMI), human urothelial cell line SV-HUC-1 (F-12), murine fibroblast NIH-3T3 (DMEM), and murine bladder cancer cell MBT2 (RPMI). Human bladder cancer MGH-U1 and its multi-drug resistant derivative, MGH-U1R, were provided by Dr. Cheng-Keng Chuang (Department of Urology, Chang Gung Memorial Hospital) [55 (link)].
All culture conditions and techniques adhered to the Bioresource Collection and Research Center (BCRC) guidelines. The culture media used, including MEM, DMEM, RPMI, and F-12, were indicated following the cell lines used. All media were prepared based on the manufacturer's recommendations and were supplemented with 10% fetal bovine serum (FBS, Biological Industries Israel Beit-Haemek Ltd., Israel), 2 mM glutamine, 1 mM sodium pyruvate, 50 U/ml of penicillin, and 50 μg/ml streptomycin. All media and supplements were purchased from Invitrogen (Grand Island, NY, USA).

Ulva lactuca was obtained from Taiwan Fertilizer Co. (Taipei, Taiwan). Chitosan (CS; molecular weight of 192 kDa, degree of deacetylation of ca. 85%) was supplied by Sigma Chemical Co. (St. Louis, MO, USA). Murine RAW 264.7 (murine macrophage), NIH 3T3 (mouse fibroblast), and HaCaT cells (human keratinocyte cells) were provided by the Bioresource Collection and Research Center (BCRC), Hsinchu, Taiwan. The culture medium for NIH 3T3 and HaCaT cells was DMEM/High glucose medium (Gibco^®, Thermo Fisher Scientific Inc., Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS), 3.7 g of sodium bicarbonate and antibiotics (100 U/mL Penicillin and 100 μg/mL Streptomycin), whereas the medium for culturing RAW 264.7 was also DMEM/High glucose medium yet supplemented with 10% FBS, 2.5 g of sodium bicarbonate, 3.7 g of HEPES and antibiotics (100 U/mL of Penicillin and 100 μg/mL of Streptomycin). All other chemicals were of reagent grade and purchased from Sigma Chemical Co., unless stated otherwise.