TLR4 Inhibition in Human ONH LC Cells

Primary human ONH LC cells were grown to confluency and pretreated with the selective TLR4 inhibitor, TAK-242 (In vivoGen, San Diego, CA, USA) at 15mM for 2 hours. TAK-242 is a cyclohexene derivative that specifically inhibits TLR4 signaling by binding to the intracellular domain of TLR4 and blocking downstream signaling. We previously performed a dose response curve using TAK-242 in primary human trabecular meshwork cells in culture and determined 15μM had the greatest inhibitory effect without affecting cell viability (29 (link)). Cells were then treated with TGFβ2 (5ng/mL) and/or TAK-242 (15μM) for 72 hours in serum-free medium. For TLR4 activation studies, hONH LC cultures were grown to confluency and treated with cellular fibronectin (cFN) (10μg/mL) containing the FN-EDA isoform (F2518; Sigma-Aldrich Corp., St. Louis, MO, USA) and/or TGFβ2 (5ng/mL), and/or TAK-242 (15μM) for 72 hours in serum-free medium. Western blot and immunocytochemistry experiments were performed as described below.