*2.2. Integrin Translocation by UDCA-LPE Suppresses FAK and SRC Phosphorylation*

Translocation of integrin β1 with subsequent loss of its co-localization with SRC (Figure 1A) was associated with decreased phosphorylation of FAK (Tyr925 and Tyr576/577) and SRC (Tyr416) upon UDCA-LPE treatment of CL48 (Figure 2A) and HHStec cells (Figure 2B) in a time-dependent manner from 15 min to 2 h. In CL48 cells, phosphorylation of c-Jun N-terminal kinases (JNK) which is a downstream target protein of FAK was also decreased by UDCA-LPE treatment (Figure 2A). Thus, UDCA-LPE inhibited integrin signalling after induction of integrin internalization via an inhibition of FAK and SRC phosphorylation.

**Figure 2.** UDCA-LPE inhibits phosphorylation of SRC and FAK. (**A**) CL48 cells and (**B**) HHStec cells were treated with 90 μM UDCA-LPE for 1 min to 2 h. Cell lysates were probed with antibodies against phospho-FAK (Tyr925), phospho-FAK (Tyr576/577), FAK, phospho-SRC (Tyr416), SRC and phospho-JNK (Thr183/Tyr185). GAPDH was used as control for equal protein loading.

#### *2.3. RGD-Containing Peptide GRGDSP Inhibits UDCA-LPE-Induced Translocation of Integrins*

The most prevalent integrin recognition site present in ECM contains a tripeptide motif composed of L-arginine, glycine and L-aspartic acid (RGD). RGD-containing peptides, which bind to the RGD-recognition site of integrin, inhibit their binding to the ECM. Although UDCA-LPE mediated the translocation of multiple integrins, it did not induce the translocation of integrin α1 as observed in CL48 cells (Figure S5). Integrin α1 can uniquely form a α1β1 heterodimer, which unlike most other integrins does not recognize RGD motif in ECM [15]. The lack of integrin α1 translocation implies that UDCA-LPE-induced translocation of integrins may solely depend on the RGD-recognition motif. GRGDSP peptide which blocks the RGD-recognition motif in integrins was therefore used to disrupt the binding of integrins to the RGD motif in ECM. GRGDSP alone had no effect on integrin localization (Figure 3). However, pre-treatment with GRGDSP markedly blocked UDCA-LPE-induced translocation of integrin β1 (Figure 3).

**Figure 3.** RGD-containing peptide GRGDSP inhibits UDCA-LPE–induced translocation of integrins. Representative fluorescence microscopy images of CL48 cells after treatment with 200 μg/mL RGD-containing peptide GRGDSP for 1 h and 90 μM UDCA-LPE for additional 1 h. IF staining of integrin β1 (green), SRC (red) and DAPI (blue). DAPI was used for nuclear staining.

#### *2.4. UDCA-LPE Binds to Integrins with Its UDCA-Moiety*

It is known that activation of integrin signalling involves autophosphorylation of FAK at Tyr397, which leads to an interaction of FAK with SRC [12]. With a short incubation time of 1–5 min, UDCA-LPE stimulated the phosphorylation of FAK (Tyr397) as well as the downstream targets c-Raf (p-Ser338) and ERK (p-Thr202/Tyr204) (Figure 4A). This activation was inhibited by GRGDSP pre-treatment. Phosphorylation of FAK at Tyr397, c-Raf and ERK was also observed by UDCA treatment (Figure 4B). Similar to GRGDSP, pre-treatment with FAK inhibitor 1,2,4,5-benzenetetraamine tetrahydrochloride (Y15) significantly blocked UDCA-LPE-induced phosphorylation of FAK (Tyr397), c-Raf and ERK (Figure 4C), suggesting a FAK-dependent mechanism. We found that RGD peptide alone also induced the phosphorylation of c-Raf and ERK after 1-5 min treatment time (Figure 4D) in a similar manner as UDCA-LPE (Figure 4D) and UDCA (Figure 4B). This suggested that these compounds triggered

integrin signalling in a similar manner like RGD peptide. Taken together, our results suggest that an interaction of UDCA-LPE with integrins may employ the UDCA-moiety of the molecule. Further binding experiments have to prove this hypothesis.

**Figure 4.** UDCA-LPE and UDCA induce integrin-dependent phosphorylation of c-Raf and ERK. (**A**–**C**) CL48 cells were treated with (**A**,**B**) 100 μg/mL RGD containing peptide GRGDSP or (**C**) 100 μM FAK inhibitor 1,2,4,5-benzenetetraamine tetrahydrochloride (Y15) for 30 min and (**A**,**C**) 90 μM UDCA-LPE or (**B**) 90 μM UDCA for 1 to 15 min. Lysates were probed with antibodies against phospho-FAK (Tyr397), phospho-c-Raf (Ser338) and phospho-ERK 1/2 (Thr202/Tyr204). (**D**) CL48 cells were treated with 100 μg/mL RGD peptide or 90 μM UDCA-LPE for 1 min to 2 h. Lysates were probed with antibodies against phospho-c-Raf (Ser338) and phospho-ERK 1/2 (Thr202/Tyr204). GAPDH was used as control for equal protein loading.
