Propidium iodide staining solution

In order to understand the effect of compound 6 and compound 14 on cancer cells, we performed a cell cycle analysis. Both tumor cell lines were seeded on 6 cm dishes so as to achieve 50% confluence. After 24 h, cells were treated at a 1 µM concentration of compound 6 and compound 14 at different time points. After that, supernatant was aspired and discarded and cells were detached using trypsin and collected after having been centrifuged at 3000 rpm for five minutes. Then, the pellet was washed with cold PBS. Consequently, cells were fixed with 70% of cold ethanol on a vortex so as to break apart a cluster of cells and incubated o/n at −20 °C. Later, the pellet was resuspended in 0.4 mL propidium iodide staining solution (4ABiotech, Co., Ltd, Beijing, China) and incubated for 30 min at RT. Then, samples were acquired using BD FACS CANTO II and the next analysis was performed using Modfit LT^TM software.

Six-well plates (Corning, New York, United States) were seeded with 16-HBE cells at a concentration of 2 × 10⁵ cells per well. At 24 h after seeding, cells were treated with vehicle or test compounds and incubated for 24 h. Cells were then digested with trypsin to prepare single-cell suspensions. The digests were centrifuged and resuspended in antibody-binding buffer, then the cells were counted. Annexin V-FITC (4A biotech, Suzhou) (5 mL per sample) was added to each sample of 1 × 10⁵ resuspended cells and mixed gently. Samples were incubated for 10 min in the dark at room temperature. After centrifugation, the supernatant was discarded, and cells were resuspended in 100 mL of antibody binding buffer. Propidium iodide staining solution (4A biotech, Suzhou) (10 mL) was added to each sample and gently mixed well, then staining was immediately halted by adding PBS. Flow cytometry was performed immediately (Beckman Coulter, United States).