Modulation of hTM Cell Signaling

Primary hTM cells were cultured on plastic dishes or glass coverslips to confluency (approximately 90%) in 10% fetal bovine serum growth media. Cells were subsequently serum starved for 24 hours, after which respective treatment with vehicle control (veh), LPA (20 µM; catalog number: 10010093; Cayman Chemical, Ann Arbor, MI, USA), IL6 (100 ng/mL; catalog number: SRP3096; Sigma Aldrich, St. Louis, MO, USA)/sIL6R (200 ng/mL; catalog number: SRP3097; Sigma Aldrich, St. Louis, MO, USA), or both (LPA + IL6/sIL6R) in serum-free media was done for 24 hours. In another set of experiments, the aforementioned treatments were performed in the presence or absence of 2 µM verteporfin (YAP inhibitor, without light stimulation using aluminum foil; catalog number: 17334; Cayman Chemical, Ann Arbor, MI, USA) or 2 µM STAT3 inhibitor (Catalog number: 573097; Sigma Aldrich, St. Louis, MO, USA) in serum-free media for 24 hours. The concentration of verteporfin used in this study has previously been verified to be safe and efficacious in hTM cells and other ocular/nonocular cells.⁵⁷ (link)^–59 (link) Herein, we determined a safe and effective dose for the STAT3 inhibitor (that is, 2 µM) by performing a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (Supplementary Fig. S1) and Western blotting.