*2.8. ET-1 ELISA*

For analysis of ET-1 secretion from LSECs, cells were kept in culture after isolation as described for 24 h. After washing of cells with PBS, they were treated with serum-free medium containing DMSO, 10 μM TGR5 agonist, 25 μM TLC, or 10 μM forskolin for 24 h. Then, the supernatant was collected and transferred onto amicon ultra-10 centrifugal filters (Merck Millipore, Burlington, NJ, USA) to concentrate the supernatant (1:10). The concentrated supernatant was analyzed for ET-1 levels using the Quantikine kit system for murine ET-1 (Research and Diagnostic Systems Inc, Minneapolis, MN, USA), according to the provided protocol. LSECs were washed and incubated with a fixed amount of

50 μL lysis buffer for protein isolation. Measurement of protein concentration in lysate was performed in the Multiskan Spectrum (Thermo Fisher) and ET-1 concentration in the supernatant was normalized relative to protein concentration.
