Immunohistochemical staining

Tissue samples were soaked in 10% neutral buffered formalin, embedded in conventional paraffin, and the section thickness was ~3 µm. Following deparaffinization, the specimens were hydrated and incubated with an epitope retrieval solution (Boster, Co., Ltd.; pH 6.0) in a microwave (temperature controlled at 95–100°C) for 20 min. The slices were then cooled to room temperature and incubated with 0.3% H₂O₂ for 10 min at room temperature to inactivate endogenous peroxidase, and then rinsed with PBS. The specimens were then incubated with rabbit polyclonal antibody for Bmi-1 (cat. no., ab85688; dilution, 1:500; Abcam, Cambridge, UK) and mouse monoclonal antibody for PADI4 (cat. no., ab128086; dilution, 1:500; Abcam) at 4°C overnight. Specimens were then incubated with ready-to-use secondary antibody EliVision™ plus kit (cat. no., KIT-9901; Maixin-Bio; Lab Vision, Kalamazoo, USA), according to the manufacturer's protocol. The specimens were then washed using PBS, and diaminobenzidine (Dako; Agilent Technologies, Inc., Santa Clara, CA, USA) chromogenic reagent was added. Termination of the chromogenic reaction was achieved with water. Following counterstaining with hematoxylin, specimens were dehydrated, mounted and observed under a Nikon 50i fluorescence microscope, magnification, ×200 (Nikon Corporation, Tokyo, Japan).