siRNA was prepared by Qiagen (HP GenomeWide siRNA, Qiagen, Hilden, Germany) targeting the β1 integrin sequence 5′-AAA AGT CTT GGA ACA GAT CTG-3′. Cells were washed three times with serum-free Dulbecco’s modified Eagle’s medium Nutrimix F12 + 0,5% geneticin followed

by siRNA transfection using HiPerFect transfection reagent (Qiagen) according selleck inhibitor to the manufacturer’s instruction. Results from at least three independent experiments are expressed as means ± SEM (standard error of the mean). Results were analyzed using Student’s t test: P < 0.05 was considered statistically significant. A detailed description of how starting structures for MD simulations of α5β1 integrin bound to either TUDC, TC, or GRGDSP were generated and how MD simulations of in total 1.050 μs length of these systems were performed and analyzed is provided in the Supporting Text. Integrin sequence numbers are according

to Uniprot. In isolated perfused rat liver, addition of TUDC at a concentration of 20 μmol/L induced within 1 minute the appearance of the active conformation of β1 integrin, whereas in the absence of TUDC the active β1-isoform was only scarcely detectable (Fig. 1A; see Supporting Fig. 1 for total α5β1 integrin staining). TUDC-induced β1 integrin activation was predominantly observed inside the hepatocytes (Fig. 1B). Equimolar concentrations of other bile acids, such as taurocholic acid (TC), glycochenodeoxycholic acid (GCDC), taurochenodeoxycholic acid (TCDC), or tauro-lithocholic acid 3-sulfate (TLCS) were

ineffective with regard to β1 integrin activation (Supporting Fig. 2). Rho https://www.selleckchem.com/products/VX-770.html None of the bile acids had any effect on the immunostaining for total α5β1 integrins (Supporting Fig. 3). TUDC-induced integrin activation was sensitive to inhibition by the RGD-motif containing hexapeptide GRGDSP, which also prevented swelling-induced integrin activation,14, 15 whereas the control hexapeptide GRADSP was ineffective (Fig. 1A). In line with previous data,12 TUDC induced within 1 minute phosphorylation of extracellular signal regulated kinases Erk-1/-2, which was abolished in the presence of the RGD-motif containing hexapeptide but not in presence of the inactive control hexapeptide (Fig. 2). TUDC also induced activation of the epidermal growth factor receptor (EGFR) in an RGD-hexapeptide-sensitive way (Fig. 2). TUDC-induced EGFR tyrosine phosphorylation involved tyrosine residues 845 and 1173, but not Tyr1045 (Fig. 2). Tyr845 is a known Src kinase target, which triggers an activating autophosphorylation at Tyr1173.28, 29 A similar EGFR phosphorylation pattern is induced by hypoosmotic hepatocyte swelling,30 a condition in which α5β1-integrins act as osmosensors. Swelling-induced β1-integrin activation largely occurred in the plasma membrane12 (Fig. 1B), in line with the concept that β1 integrins in the plasma membrane serve as osmo-/mechanosensors through a swelling-induced attachment to extracellular matrix proteins.