Notch Inhibitor Screening in Cells and Zebrafish

Natural compounds (#L1400) and FDA-approved chemicals (#L1300), including triptolide and DAPT for the Notch inhibitor screening test, were purchased from Selleckchem, (Houston, TX, USA). DMSO was purchased from Corning (USA, Manassas, VA, USA). Pyridone 6 (P6) was purchased from R&D Systems (a Bio-Techne Brand, Minneapolis, MN, USA). In cell-based experiments, P6, DAPT, P6 + DAPT, TP, or DMSO were treated for a selective concentration of 50 nM, 100 nM, 200 nM, 500 nM. The concentrations of each signal inhibitor and TP were determined through referencing their use in various cells [39 (link),40 (link),41 (link)] and zebrafish [74 (link),75 (link)]. The concentration ranges typically used in cell-based experiments to test anticancer agents was applied [76 (link)]. They were incubated at 37 °C, 5% CO₂, for 24 h. To reduce error, chemicals were diluted with Opti-MEM (Welgene, Republic of Korea) before suspension. In zebrafish experiments, 24 hpf embryos were placed in 90ϕ Petri dishes filled with 10 mL of E3 buffer (with 0.001 M methylene blue). Embryos were treated with DMSO for the negative control or a 500 nM concentration of P6, DAPT, P6 + DAPT, or triptolide. They were incubated for 72 h in an incubator at 28.5 °C.