*2.6. LPE-Moiety is Necessary for UDCA-LPE-Induced Translocation of Integrin β1 and Suppressed FAK and SRC Phosphorylation*

To dissect the role of LPAR1, we further utilized an LPAR antagonist Ki16425, which was reported to disrupt the binding of LPE to LPAR1 [16]. We found that Ki16425 pre-treatment significantly blocked UDCA-LPE-induced translocation of integrin β1 in a concentration-dependent manner (Figure 6A). Additionally, UDCA-LPE-induced inhibition of phosphorylation of FAK (Tyr576/577 and Tyr925) (Figure 6B) and SRC (Tyr416) (Figure 6C) was nearly completely abolished by pre-incubation with Ki16425. It has been reported that the activity of lysophosphatidic acids to bind and activate LPAR decreases with a shorter fatty-acid chain length [19,20]. Thus, we treated CL48 cells with UDCA-PE (a conjugate of UDCA and 18:1, 18:1 PE), UDCA-LPE (12:0) (UDCA conjugated with 12:0 LPE) or UDCA-LPE (14:0) (UDCA conjugated with 14:0 LPE). Unlike UDCA-LPE (UDCA conjugated with 18:1 LPE), UDCA-PE, UDCA-LPE (12:0) or UDCA-LPE (14:0) did not decrease but rather slightly increase the phosphorylation of FAK (Tyr 925 and Tyr576/577) and SRC (Tyr416), which was found to be similar to UDCA and tauro-UDCA (TUDCA) (Figure 6D). Taken together, our results suggested that the LPE-moiety and its association with LPAR1 were essential for UDCA-LPE-induced translocation of integrin β1 and inhibition of SRC and FAK phosphorylation.

**Figure 6.** Translocation of integrin β1 and dephosphorylation of SRC and FAK is dependent on the LPE moiety of UDCA-LPE. (**A**) Representative fluorescence microscopy images of CL48 cells after treatment with LPAR antagonist Ki16425 at 100 μM or 500 μM for 1 h and 90 μM UDCA-LPE for additional 1 h. IF of anti-integrin β1 (green). (**B**) CL48 cells were treated with 50 μM Ki16425 for 1 h, followed with 90 μM UDCA-LPE for additional 1h. Lysates were probed with antibodies against phospho-FAK (Tyr925) and phospho-FAK (Tyr576/577). (**C**) CL48 cells were treated with 1mM Ki16425 for 1 h, followed with 90 μM UDCA-LPE for additional 1 h. Lysates were probed with antibodies against phospho-SRC (Tyr416). (**D**) CL48 cells were treated with 90 μM UDCA, TUDCA, UDCA-LPE, UDCA-LPE-Na, UDCA-PE, UDCA-LPE 12:0 or UDCA-LPE 14:0 for 2 h. Lysates were probed with antibodies against phospho-FAK (Tyr925) and phospho-FAK (Tyr576/577). GAPDH was used as control for equal protein loading.
