1 oleoyl lysophosphatidic acid lpa

Male 6-8 weeks old wild-type (WT) mice (Jackson Laboratory, Bar Harbor, ME) or Nrf2-deficient mice (breeding pairs provided by Dr. Thomas Kensler, Johns Hopkins University) on a C57BL/6 background (backcrossed for at least 12 generations) were used in a model of partial warm hepatic IR, as described^{1 (link)}“^{3 (link)}. In general, the arterial/portal vessels to the cephalad lobes were clamped for 90min. No vascular occlusion in sham-controlled mice. In the treatment groups, animals were infused at 1h prior to the onset of liver ischemia with a single dose of YAP activator (1-oleoyl lysophosphatidic acid (LPA): 0.8μmol/kg i.v., Tocris Bioscience, Minneapolis, MN) or YAP inhibitor (verteporfin (VP): 0.8pmol/kg i.v., MilliporeSigma, St. Louis, MO) dissolved in PBS. Mice were sacrificed at various time-points after reperfusion; liver and serum samples were collected for analysis. All animal experiments were approved by UCLA Animal Research Committee.

After in situ collagenase digestion, mouse primary hepatocytes were separated by Percoll density centrifugation, and cultured for 24h. After pretreatment with PACAP (10nM), VP (20nM), 1-oleoyl lysophosphatidic acid (LPA, 100nM, Tocris Bioscience, Minneapolis, MN), H89 (10μM), or CREB inhibitor (10μM) for 1h, (PBS or DMSO as vehicle control), hepatocyte necrosis was triggered by hydrogen peroxide (H₂O₂, 0.1mM, Sigma-Aldrich).