Triton x 100

Proliferation of the cells was examined using a BeyoClick™ EdU-647 kit (Beyotime). In short, the cells were sorted in 6-well plates. After adherence, the medium was renewed, and each well was loaded with 10 μM EdU solution and the cells were incubated at 37℃ for 2.5 h. Next, the cells were fixed in 4% PFA (Beyotime) for 15 min, permeabilized in 0.3% Triton X-100 (Elabscience Biotechnology Co., Ltd., Wuhan, Hubei, China) for 8 min, and then added with 500 μL Apollo staining buffer in the dark for 40 min. The nuclei were stained with 4', 6-diamidino-2-phenylindole (DAPI) for 10 min. The staining was observed under a fluorescence microscope (Zeiss, Oberkochen, Germany) and quantified using the Image J software.

PriGIST cells in the logarithmic growth phase were seeded at a density of 2 × 10⁴ cells/well in a 12-well plate with preplaced crawl sheets. Immunofluorescence staining was performed at 80% cell density after approximately 12 h of culture. Cells were fixed in 4% paraformaldehyde (Elabscience, Wuhan, China) for 30 min and washed three times in PBS (Procell, Wuhan, China). The cells were permeabilized with 0.1% Triton X-100 (Elabscience) for 20 min and then incubated in 5% bovine serum albumin (BSA; Elabscience) for 2 h to block the antigen. Primary antibodies for DOG-1 (1:100 dilution, ZSGB-BIO, Beijing, China), CD34 (1:100 dilution, ZSGB-BIO), and CD117 (1:100 dilution, ZSGB-BIO) were added and incubated overnight at 4 °C. Goat anti-rabbit IgG (H + L) (1:100 dilution; Elabscience) was used as the secondary antibody. The cell nuclei were re-stained with 4′,6-diamidino-2-phenylindole (DAPI, Solarbio). Images were captured using a confocal microscope (Olympus Corporation, Tokyo, Japan).

Hep3B and Huh1 cells were seeded onto Poly-lysine (P4707, Millipore Sigma, Darmstadt, Germany) coated cover glasses. 1–2 days later, cells were washed with PBS three times at room temperature and then fixed with 4% paraformaldehyde Fix Solution (Cat: E672002, Sangon, Shanghai, China) for 15 min. Next, the fixed cells were permeabilized with 0.2% Triton X-100 (Cat: E-IR-R122, Elabscience Biotechnology, Wuhan, China) for 10 min at room temperature. Sections were incubated with Ki67 (Cat: 27,309–1-AP, Proteintech, Rosemont, USA) or β-catenin (Cat: #8480, CST) primary antibodies at 4ºC overnight. The next day, sections were washed using PBS for 3 × 5 min at room temperature, and then incubated with 4',6-diamidino-2-phenylindole (DAPI) dye (Cat: D1306, ThermoFisher Scientific, Waltham, MA, USA) and fluorophore-conjugated secondary antibodies at room temperature for 2 h. Images were captured by the Leica microscope.