Mitomycin mmc

HeLa cells and KGN cells were cultured in 24-well plates and transiently transfected with wild-type, mutant, and polymorphic HSF2BP-FLAG plasmids with the use of Lipofectamine 3000 (Invitrogen, Carlsbad, CA, United States). After being cultured for 48 h, the cells were divided into three groups: without treatment, treated with etoposide (ETO; 5 μg/ml, Solarbio, Beijing, China) for 1 h, and treated with mitomycin (MMC; 500 ng/ml, Sigma-Aldrich, St. Louis, MO, United States) for 8 h. Then, the treated cells were cultured with a normal medium and recovered for a specific time at 37°C. Then, the cells were fixed with 4% paraformaldehyde (Solarbio) for 20 min at room temperature. Then the slides were permeabilized with 0.3% Triton X-100, blocked with 10% bovine albumin for 1 h, incubated with anti-FLAG antibody (1:500 dilution, CST14793, Cell Signaling Technology, Danvers, MA, United States) at 4°C overnight, followed with incubation with fluorescent secondary antibody for 1 h, and then treated with antifade mounting medium with DAPI (Beyotime, Shanghai, China). Intervening phosphate-buffered saline (PBS) washes were performed after incubation when necessary. Images were captured with an Olympus (Tokyo, Japan) BX61 microscope. The nuclear vs. cytoplasmic ratio of the HSF2BP proteins in HeLa cells and KGN cells was quantified by ImageJ software.