Cell cycle assay

H1975, HCC827, A549, and H1299 human NSCLC adenocarcinoma cells were obtained from the American Type Culture Collection. A431 human epidermoid carcinoma cells were kind gifts from Fuheng Biology Company (Shanghai, China). LoVo human colon carcinoma cells, HBE human bronchial epithelioid cells, LO-2 human normal hepatocyte cells were kind gifts from YuXi Biotech Company (Jiangsu Province, China). The Cell Counting Kit-8 (CCK-8) reagent was obtained from Biotool Company (Kirchberg, Switzerland). The wild-type EGFR enzyme, mutant EGFR L858R/T790M enzyme, and the ADP-Glo™ Kinase Assay system that measures ADP formed from a kinase reaction were purchased from Promega Corporation (Fitchburg, WI, USA). The Annexin V-FITC Apoptosis Detection Kit and Cell Cycle Assay were purchased from Beyotime Company (Shanghai, China). H1975, HCC827, and H1299 cells were grown in RPMI-1640 (Gibco^®, Big Cabin, OK, USA) supplemented with 10% FBS (Gibco^®) and 1% penicillin-streptomycin (Beyotime Company, Shanghai, China). A431, LoVo, HBE, and LO-2 cells were grown in DMEM (Gibco^®) supplemented with 10% FBS (Gibco^®) and 1% penicillin-streptomycin (Beyotime Company, Shanghai, China). All cells were maintained and propagated as monolayer cultures at 37 °C in humidified 5% CO₂ incubator.

36 hours after siRNA transfection, cells were plated into 24-well plates at the concentration of 50000 cells per well. 24 hours after seeding, cells were collected and divided into two parts. One part was used for cell apoptosis assay. Briefly, cells were stained with propidium iodide (PI) and Annexin V-FITC following the manufacturer's instructions (KeyGEN, Jiangsu, China). Flow cytometer was used to detect cell apoptosis. Another part was used for cell cycle assay (Beyotime, Shanghai, China). Collecting cells were rinsed in ice-cold PBS before being fixed in 75% methanol over the night at 4°C. Following that, cells were stained for 20 minutes with PI, RNase A, and Triton X-100. Cell apoptosis assay and cell cycle assay were performed using flow cytometry.