*4.1. Reagents and Cell Culture*

All reagents as well as the cultures and treatment of human embryonic liver CL48 cell line and Human Hepatic Stellate Cells (HHStec) are shown in Supplementary Materials.

#### *4.2. Western Blotting*

Lysates of treated cells were subjected to western blotting analysis (Supplementary Materials).

#### *4.3. Immunofluorescence*

Paraformaldehyde-fixed cells were subjected to immunofluorescence (Supplementary Materials).

#### *4.4. Lipid Fractionation*

For each treatment group, CL48 cells were cultured in 20 × 75 cm2 culture flasks. After UDCA, LPE or UDCA-LPE treatment, cells were rinsed with PBS and scraped into 10 mL buffer containing 2 mM HEPES, 150 mM NaCl, 1 mM EGTA, 5 mM sodium vanadate, 10 mM sodium azide, 10 mM sodium pyrophosphate, 100 μg/mL PMSF, 1 mM sodium orthovanadate and 10 μl/mL protease inhibitor cocktail. Cells were homogenized and the lysates were centrifuged at 800× *g* at 4 ◦C for 10 min. Two mL of supernatants were incubated at 37 ◦C for 4 min and then incubated with 0.02 g Brij 98 at 37 ◦C for 5 min. The extracts were adjusted to 4 mL with 2 M Sucrose and cooled down in ice for 1 h. The extracts were gently

overlaid with successive decreasing sucrose densities solutions (0.9–0.8–0.75–0.7–0.6–0.5–0.4–0.2 mol/L Sucrose) to prepare a discontinuous sucrose gradient. The gradients were centrifuged at 200,000× *g* in a Beckman SW 41Ti rotor for 22 h at 4 ◦C. Twelve fractions (1 mL for each fraction) were collected and used for western blotting and liquid-chromatography mass spectrometry (LC/MS-MS) analyses. The concentrations of total targets (proteins or lipids) in 12 fractions were normalized to 100% and the proportion or abundance of each target was reported in %. Cell lysates were subjected to sucrose density-gradient centrifugation for lipid fractionation.

## *4.5. Quantification of UDCA-LPE, UDCA and LPE*

Following lipid fractionation, 500 μL of each fraction were extracted with 3 mL chloroform-methanol 2:1 mixture, 500 μL water and 20 μL internal standard D4-UDCA. Following centrifugation at 2500 rpm for 5 min, the lower chloroform phase was collected. Three mL of 2:1 chloroform-methanol mixture was added to the upper phase, extracted the second time and again centrifuged at 2500 rpm for 5 min. The lower phase was collected, combined with the previous chloroform phase and added to 0.4 mL 50 mM citric acid. Following mixing and centrifugation the lower phase was collected in a glass tube and the solvent was evaporated to dryness. The dried lipids were dissolved in 180 μL methanol. Concentrations of UDCA-LPE, UDCA and LPE in each lipid fraction were quantified using a liquid-chromatography mass spectrometer. The responses were calculated from the ratio of UDCA-LPE, UDCA, or LPE peak and D4-UDCA. Concentrations in nmol/mg protein were calculated from response of UDCA-LPE, UDCA and LPE used in standard curves. LC/MS-MS machine and running conditions are described in our published work [44]. Briefly, the separation was achieved by using a Phenomenex Luna C18 (Phenomenex, Aschaffenburg, Germany) column (100 × 2.0 mm, 3 μm) fitted on a separation module of a Waters 2695 (Waters, Milford, MA, USA). Binary solvents were 80% H2O/MeOH with 8 mM ammonium acetate, pH 8.0 (solvent A) and 95% MeOH/H2O with 8 mM ammonium acetate, pH 8.0 (solvent B). The flow rate was maintained at 0.2 mL/min and the gradient was started with 100% solvent A for 2.5 min, changed to 100% solvent B in 1 min, held for 16.5 min and returned to the initial condition in 3 min. Separated fractions were detected on-line by an electrospray ionization source of the tandem mass spectrometer (Quattro micro API, Micromass Waters, Waters, Milford, MA, USA).

#### *4.6. Immunoprecipitation*

Lysates of treated cells were subjected to immunoprecipitation analysis (Supplementary Materials).

#### *4.7. Statistical Analysis*

Statistical analysis was performed using Prism Software version 4.0 (GraphPad, La Jolla, San Diego, CA, USA).

Please see Supplementary Materials for detailed information.

#### **5. Conclusions**

UDCA-LPE enforces internalization of integrins leading to an inhibition of downstream signalling pathways. As a possible novel mode of integrin inhibition, we described the simultaneous bivalent ligation of integrins and LPAR1 by via the LPE endocytic transport pathway. Thus, UDCA-LPE emerges as drug candidate for treatment of liver fibrosis by inhibiting integrin signalling via its internalization.

#### **Supplementary Materials:** Supplementary materials can be found at http://www.mdpi.com/1422-0067/19/10/ 3254/s1.

**Author Contributions:** Conceptualization, J.S. and A.P.; Formal analysis, J.S.; Funding acquisition, A.P.; Investigation, J.S.; Methodology, J.S., H.G.-S. and B.G.; Resources, W.S.; Supervision, A.P.; Validation, W.C. and A.P.; Writing–original draft, J.S.; Writing–review & editing, W.C., W.S. and A.P.

**Funding:** This study was supported by the German Research Foundation (PA 2365/1-1). A.P. was funded by the Olympia Morata Postdoctoral Fellowship of the Medical Faculty of University of Heidelberg.

**Acknowledgments:** The authors thank Fortunata Jung for excellent technical assistance. We acknowledge financial support by Deutsche Forschungsgemeinschaft within the funding programme Open Access Publishing, by the Baden-Württemberg Ministry of Science, Research and the Arts and by Ruprecht-Karls-Universität Heidelberg.

**Conflicts of Interest:** The authors who have taken part in this study declare that they do not have anything to disclose regarding funding or conflict of interests. W.S. has a patent on UDCA-LPE (no industrial funding).
