Apc annexin 5 assay

Cell proliferation was measured using CellTiter 96 Aqueous One solution assay (Promega Corporation, Madison, WI) according to the manufacturer's directions as described previously [44 (link), 47 (link)]. Cellular apoptosis was measured using APC-Annexin-V assay (BD Biosciences, San Diego, CA). Levels of p62 were measured using p62 ELISA kit according to the manufacturer's instructions (Enzo life Science, Farmingdale, NY). Immunoblot analysis and Real-Time PCR were conducted as previously described [24 , 44 (link), 47 (link)]. Primers used in real-time PCR are shown in table 2.

Cytotoxicity was monitored using the LDH-Glo™ cytotoxicity assay (Promega, Madison, WI, United States). Briefly, cells were plated in 96-well plates at a density of 5,000 cells and treated with EGFR-TKIs for 24 h. Supernatants were incubated with the reagent for 60 min in the dark. After incubation, luminescence for cell cytotoxicity was measured using a GloMax NAVIGATOR (Promega). All experiments were performed in triplicate.
Induction of apoptosis by EGFR-TKIs was assessed using the APC-Annexin V assay according to the manufacturer’s instructions (BD Biosciences, United States). Additionally, 7-Amino-Actinomycin D (7-AAD) (BD Biosciences) was added to the binding buffer to stain the necrotic and dead cells. The resuspended cells in the stain solution were gently vortexed and incubated for 15 min at room temperature in the dark. Stained cells were detected using a flow cytometer (BD FACSCalibur™, Becton Dickinson, United Kingdom) and analyzed using the FlowJo v10.7.2 system.