2.5. Immunofluorescent staining

To examine the protein expression by immunofluorescent staining, lung cancer cells were seeded onto coverslips in a 24-well plate and left overnight. Cells were then fixed using 4% formaldehyde for 30 min at 25 °C and treated with 2% bovine serum albumin (BSA) in phosphate buffered saline (PBS) for 30 min. The coverslips were incubated with rabbit anti-UBE2C, Ki67, Annexin V, ABCG2, ERCC1, Vimentin and mouse anti-E-cadherin monoclonal antibody (Abcam) at 1:200 dilution in 3% BSA. The coverslips were then incubated with an Alexa-Fluor 467 (green, 1:500, A-11029; Invitrogen, USA) and 594 (red, 1:500, A-11032; Invitrogen, USA) tagged anti-rabbit or anti-mouse monoclonal secondary antibody at 1:1000 dilution in 3% BSA. Hoechst (3 μg/ml, (cat. no. E607328; Sangon Biotech Co., Ltd.) was added for nuclear counterstaining. Images were obtained with a Zeiss Axio Imager Z1 Fluorescent Microscope (Zeiss, Oberkochen, Germany).