Stable Cell Lines for Fluorescent Fusion Proteins

Cells were plated in a six-well plate (Thermo Fisher Scientific) 1 day before the transfection of the expression constructs for mCherry-tagged proliferating-cell nuclear antigen (PCNA; Leonhardt et al., 2000 (link)) or EGFP-ATM based on PB533A-2 (System Biosciences) with a PiggyBac transposon expression vector (PB210PA-1; System Biosciences), or pEGFP-C1-FLAG-Ku80 (Addgene 46958; Britton et al., 2013 (link)) using FuGENE HD Transfection Reagent (Promega) according to the manufacturer's instruction. The EGFP-ATM expression vector was constructed using a plasmid containing ATM cDNA provided by Tsuyoshi Ikura (Kyoto University, Japan) and the entire ATM sequence was verified by sequencing. To obtain a stable cell line, 2 days after the transfection, the cells were incubated in the presence of 1 mg/ml G418 disulfate aqueous solution (Nacalai Tesque) in the complete medium for >1 week. Cells that exhibited mCherry–PCNA or EGFP–ATM fluorescence were sorted using a cell sorter (Sony; SH800) and cultured in fresh medium without G418.