Ebm mv2

Human dermal lymphatic endothelial cells (LECs) from three different donors (n = 3) were purchased from PromoCell (C-12217, PromoCell GmbH, Heidelberg, Germany). Cryopreserved cells were thawed and expanded in an expansion medium, which consists of Endothelial Cell Basal Medium MV2 (EBM MV2, PromoCell GmbH, Heidelberg, Germany) supplemented with SupplementMix (C-39226, PromoCell, Heidelberg, Germany), containing a company-stated optimal formula of 5% fetal calf serum (FCS), 5 ng/mL epidermal growth factor (EGF), 10 ng/mL basic fibroblast growth factor (bFGF), 20 ng/mL insulin-like growth factor (IGF), 0.5 ng/mL vascular endothelial growth factor 165 (VEGF-A), 1 μg/mL ascorbic acid, and 0.2 μg/mL hydrocortisone, and were incubated at 37 °C and 5% CO₂. Ensuing experiments were all carried out between the 3rd–5th cellular passages. The negative control (=basal) media consisted of Endothelial Cell Basal Medium MV2 (EBM MV2, PromoCell GmbH, Heidelberg, Germany) supplemented with 1 μg/mL ascorbic acid, 0.2 μg/mL hydrocortisone, and 1% FCS from the SupplementPack (C-39221, PromoCell, Heidelberg, Germany). For the positive control, we used the expansion media tested and proven by the company to be optimal for LECs. HPS and NS were used as undiluted (100%) and diluted with basal media at 0.1%, 1%, 10%, and 40% final concentrations for the following experiments. PRP was not diluted.

Human dermal lymphatic endothelial cells (HLECs) from three different de-identified donors were purchased from Promocell (Heidelberg, Germany) and were maintained in complete endothelial basal media (EBM-MV2, Promocell, Heidelberg, Germany) at 37 °C with 5% CO₂. The cell characteristics were verified by the expression of the HLEC specific markers, namely LYVE1, PROX1, and podoplanin (PDPN), as described previously [25 (link)]. The TGR5 specific inhibitor SBI-115(3-methylphenyl 5-chloro-2-(ethylsulfonyl)-4-pyrimidinecarboxylate) was purchased from Sigma Aldrich (St. Louis, MO, USA). The RSK inhibitor BI-D1870 was purchased from Selleckchem (Houston, TX, USA). The YAP inhibitor verteporfin was purchased from Cayman Chemical (Ann Arbor, MI, USA) and the N-Acetyl-L-cysteine (NAC), the specific ROS inhibitor, was purchased from Sigma Aldrich (St. Louis, MO, USA). For the inhibitor treatments, HLECs were pre-treated with SBI-115 (10 µM), BI-D1870 (1 µM), NAC (10 mM), or verteporfin (0.5 µM) 1 h prior to the BA treatment. The cell permeable TGR5 receptor agonist (phenoxypyrimidine carboxamide derivative) was purchased from Sigma Aldrich. For the RNA and protein isolation from BAs-treated HLECs, 50–150 µM of conjugated BAs were used to treat HLECs for 24 h.