Human epidermal growth factor (hegf)

Breast cell lines MDA-MB-231, BT-549, MCF-7, T47D, MCF-10A, and MCF-12A (American Type Culture Collection, MD, USA) were used. Breast tumor cells lines were routinely maintained in RPMI 1640 for tumor cell lines supplemented with 10% FBS, 2mM L-glutamine, and gentamicin. MEGM (Mammary Epithelial Growth Medium) was used for normal epithelial cell lines supplemented with 10% FBS, 2mM L-glutamine, and a growth factor kit containing BPE, hydrocortisone, hEGF, insulin, and gentamicin (Lonza, MD, USA) in a 37 °C incubator with 5% CO₂. Breast cancer cell lines T47D A18 and T47D A18 4OHT, WS8, and MCF7 ICI resistant cell lines were obtained from Jorden V. Craig [45 (link)]. Cells lines were routinely maintained in RPMI 1640 supplemented with 10% FBS, 2mM L-glutamine, and 0.05 mg/ml gentamicin in a 37 °C incubator with 5% CO₂. Human umbilical vein endothelial cells (HUVECs) were obtained from Lobie P.E. (CSI, NUS, Singapore). Cells lines were routinely maintained in EBM (Endothelial Basal Medium) supplemented with 10% FBS, 2mM L-glutamine, and a growth factor kit containing hEGF, hydrocortisone, ascorbic acid, VEGF, hFGF-B, IGF-1, and gentamicin (Lonza, MD, USA) in a 37 °C incubator with 5% CO₂.

Human intrahepatic CCA cell line KKU-M055 was established in the Cholangiocarcinoma Research Institute, Faculty of Medicine, Khon Kaen University. An immortalized human cholangiocyte cell line MMNK1 was used as the reference cholangiocyte. KKU-M055 and MMNK1 cells were cultured in a dulbecco's modified eagle medium (DMEM) supplemented with 1% penicillin/streptomycin (P/S) and 10% fetal bovine serum (FBS) (all from Life Technologies, Paisley, UK). HMVECs were cultured in endothelial basal medium (EBM-2) supplemented with 10% FBS, 1% P/S, 10 ng/mL human epidermal growth factor (hEGF), and 1 μg/mL hydrocortisone (all from Lonza, Walkersville, MD, USA). All cells were cultured in a humidified incubator at 37 °C with 5% CO₂.

Human pulmonary arterial smooth muscle cells (hPASMCs) isolated from healthy individuals without PAH (donors) were purchased from Lonza (Basel, Switzerland) and PASMCs from idiopathic pulmonary arterial hypertension (IPAH) patients were obtained from Giessen biobank, Justus–Liebig University, Germany. The collection of the human material was performed in accordance with the protocol approved by the ethics committee of the University Hospital Giessen (Germany) in accordance with the European IPS registry (AZ 111/08) and DZL Biobank (58/15). Tissue donation was approved by the ethics committee of the University Hospital Giessen in agreement with the general principles in the Declaration of Helsinki.
Cells were cultured in growth medium smooth muscle cell growth medium (SMGM) containing a cocktail of insulin, human fibroblast growth factor (hFGF), fetal bovine serum (FBS), and human epidermal growth factor (hEGF) obtained from Lonza (Basel, Switzerland). All experiments were performed between passages three to five in the starvation medium (SMBM, without the addition of growth factors) (Basel, Switzerland) or SMGM with or without PDGF-BB stimulation (30 ng/mL).