Wortmannin

A human RPE cell line (ARPE-19) was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). ARPE-19 cells were cultured in Dulbecco's modified Eagle's medium (DMEM)/F12 medium containing 10% fetal bovine serum in a humidified atmosphere at 37°C with 5% CO₂. The ARPE-19 cells were used at passages two to four in all the experiments and were treated with recombinant TGF-β2 (Santa Cruz Biotechnology, Santa Cruz, CAm USA) at a concentration of 10 ng/mL to simulate the PVR environment.
To disrupt the expression of EGFR, YAP, or RhoA, the ARPE-19 cells were treated with small interfering RNA (siRNA) against EGFR, YAP, or RhoA. EGFR-siRNA, YAP-siRNA, RhoA-siRNA and nonsilencing siRNA were obtained from Invitrogen (Carlsbad, CA, USA). Lipofectamine 2000 transfection reagent (ThermoFisher Scientific, Waltham, MA, USA) was used to perform transient transfection of siRNA. To disrupt EGFR signaling, ARPE-19 cells were treated with erlotinib (100 nM; Millipore) for 24 hours. To inhibit PI3K signaling, ARPE-19 cells were treated with LY294002 (25 µM; Bio-Rad Laboratories, Hercules, CA, USA) or wortmannin (100 nM; Bio-Rad Laboratories). To disrupt PDK1 signaling, ARPE-19 cells were treated with GSK2334470 (10 µM; Bio-Rad Laboratories). To disrupt Akt signaling, ARPE-19 cells were treated with MK-2206 (10 µM; Millipore).