Verteporfin

Semiconfluent cultures of human TM cells were incubated overnight in serum-free media unless otherwise noted. Then, serum-starved cultures were subjected to different VEGF concentrations (1 ρg/mL, 10 ρg/mL, 100 ρg/mL, 1 ng/mL, 10 ng/mL, and 30 ng/mL) for 48 h (Figure 8A). In the control cultures, the medium was changed at the same time points, but without VEGF. In another set of experiments, the aforementioned experiments with 30 ng/mL VEGF were performed. The cells were divided into three groups based on the subsequent treatment protocol. (1) VEGF-only group: human TM cells were untreated after VEGF exposure; (2) VEGF + Vehicle group: human TM cells were treated with the vehicle (phosphate-buffered saline; PBS) solution for 24 h after exposure to VEGF; (3) VEGF + verteporfin group: human TM cells were treated with 1 μM verteporfin (Sigma-Aldrich; St. Louis, MO, USA) for 24 h after exposure to VEGF. In the control group, the medium was changed at the same time points without VEGF, vehicle, or verteporfin exposure. The verteporfin concentration used in this study has been verified as safe and efficacious in human TM cells and other ocular cells [15 (link),51 (link)]. After the treatments, the cells were processed for Western blotting, quantitative real-time polymerase chain reaction (PCR), and immunocytochemistry (Figure 8B).

Verteporfin (#SML0534) and antibodies recognizing β-actin (#a2228), FLAG^® M2 (#F1804), glyceraldeyde-3-phosphate dehydrogenase (GAPDH, #CB1001) and 17-β-estradiol (#E1024) were purchased from Merck (Billerica, MA, USA). Antibodies recognizing YAP/TAZ (#8418), p-YAP (Ser127) (#4911), zinc finger E-box binding homeobox1 (ZEB1, #3396), Src (#2108), p-Src (Tyr416) (#2101), horseradish peroxidase-conjugated donkey anti-rabbit IgG (#7074), and horseradish peroxidase-conjugated donkey anti-mouse IgG (#7076) were purchased from Cell Signaling Technology (Danvers, MA, USA). Antibodies recognizing E-cadherin (#610181) and N-cadherin (#610920) were purchased from BD Biosciences (San Jose, CA, USA). The antibody recognizing p-YAP (Tyr357) (ab62751) was purchased from Abcam (Cambridge, United Kingdom). Antibodies recognizing Vimentin (sc-32322) was purchased from Santa Cruz biotechnology (Dallas, TX, USA). V5 Tag (#A190-120A), Alexa Fluor 488 donkey anti-mouse IgG and Alexa Fluor 568 goat anti-rabbit IgG were purchased from Thermo Fisher Scientific (Cleveland, OH, USA). Saracatinib (#S1006) and PP2 (#S7008) were purchased from Selleckchem (Houston, TX, USA). G418 was purchased from Biosesang (Gyeonggi-do, South Korea).

Verteporfin (VP, #SML0534, Merck, USA), ML-7 hydrochloride (ML7, #I2764, Merck, USA), Cytochalasin D (CD, #C8273, Merck, USA), AICAR (AI, #A9978, Merck, USA) were used as pharmacological inhibitors of Yap/Taz activity. Depending on the solubility of the substances, water, ethanol 96%, or Cyrene (dihydrolevoglucosenone, #807796, Merck, USA) [163 (link)] were used as solvents to prepare stock solutions. The stock solutions of VP were prepared with 10% (v/v) solution of Cyrene in water, ML7—with 50% (v/v) solution of ethanol in water, CD—with 100% (v/v) ethanol, and AI—with 100% (v/v) water.
A volume of 30 μL of stock solutions were pipetted onto the medium surface of each experimental vial. In a control vial, 30 μL of corresponding solvent were pipetted. The final concentrations of substances in the food media were estimated using blue food dye (Brilliant Blue FCF, Roha Dyechem Ltd., Mumbai, India) as a tracer of stock solution diffusion [164 (link)]. The obtained results demonstrated a 1:30 dilution of the stock solutions and a final concentration of VP—0.01, 0.1, 1, 10 μM, ML7—0.01, 0.1, 1 μM, CD, and AI—0.1, 1 μM.
Treatment with inhibitors was started from the first day of imago life and continued for 10 days for analysis of survival throughout the lifetime for qRT-PCR assay.

KBD-hiPS-Ch and N-hiPS-Ch were seeded in 96-well plates at a density of 1 × 10⁴ cells per well and treated with various concentrations of T-2 toxin (1, 2.5, 5, 10 ng/mL), DON (100, 250, 500, 1000 ng/mL), and 1:100 T-2+DON toxin (1 + 2.5, 2.5 + 250, 5 + 500, 10 + 1000 ng/mL) for 48 h. We used 0.1–10 μM verteporfin (Sigma, Cat.SML0534) to inhibit YAP protein expression in hiPS-Ch for 48 h. Cell viability was then tested using a cell-counting kit (CCK8, NCM Biotech, Suzhou, China). The absorbance at 450 nm was used to calculate relative viability using a microplate reader (Model 3550, Bio-Rad, Hercules, CA, USA).