Bovine serum albumin (bsa)

Cells (1×10⁶ cells/well) were plated in 6-well plates and after they had adhered, 4% paraformaldehyde was used to fix the cells at 4°C for 24 h. Subsequently, 0.5% Triton X-100 (Beyotime Institute of Biotechnology) was used to permeabilize the cells and 5% BSA (Beyotime Institute of Biotechnology) was used to block non-specific binding at room temperature for 1 h. A primary antibody was diluted with 5% BSA and incubated with the cells at 4°C overnight. The primary antibody used was an anti-Ki-67 (1:200; product code ab16667; Abcam). Next, the cells were incubated at 37°C for 1.5 h with the goat polyclonal secondary antibody to Rabbit IgG (heavy chain and light chain) Alexa Fluor^® 488 (1:3,000; cat. no. ab150077; Abcam) in a dark room for 1.5 h. Finally, the nucleus of these cells was stained with 1 mg DAPI for 10 min at room temperature (Invitrogen; Thermo Fisher Scientific, Inc.). The fluorescence was observed using a laser scanning confocal microscope (magnification, ×200; Olympus Corporation).

RIPA buffer (Beyotime, China) was used for the collection of protein samples. BCA (Beyotime, China) was used for the determination of the concentration of these protein samples. Next, SDS-PAGE gel (Beyotime, China) was used for the separation of these proteins. And the PVDF membranes (Millipore, USA) were used for the adsorption of these proteins. Next, these membranes were blocked with the BSA (Beyotime, China) for 2 h. After that, the primary antibodies were incubated with these membranes at 4°C overnight. The primary antibodies applied in this were MTBP (ABCAm, ab115529), ZEB2 (ABCAm, ab138222), CD44 (ABCAm, ab51037), CD133 (ABCAm, ab222782) and β-actin (ABCAm, ab8227). All these primary antibodies were diluted with the BSA (Beyotime, China) with the ratio (1:1000). These membranes were incubated with the second antibodies for 2 h in the second day. The second antibodies were also diluted with the BSA with the ratio (1:2000). Finally, immunoreactive signals were determined with the Pierce Western Blotting Substrate (Millipore, USA). The bands were quantified with the ImageJ software (National Institutes of Health, USA).

Cells (2.5×10⁵) were seeded into 24-well plates and covered by coverslips. Following the culturing of cells for 36 h and 7 days time intervals, they were removed and washed with PBS three times. Cells were subsequently fixed in 4% (w/v) cold paraformaldehyde at 4°C for 30 min, permeabilized in 0.5% (w/v) Triton X-100 at 37°C for 5 min and incubated in deionized water containing 3% (w/v) H₂O₂ at room temperature for 10 min. Cells were subsequently blocked using 5% bovine serum albumin (Beyotime Institute of Biotechnology, Jiangsu, China) at 37°C for 30 min. After rinsing with PBS, cells were incubated in anti-cytokeratin 18 primary antibody (1:400) buffer at 4°C overnight. Cells were incubated in an equal volume of PBS in the control group. After rinsing with PBS, cells were incubated in HRP conjugated polymer anti-rabbit IgG antibody (1:100) buffer at 37°C for 1.5 h. Subsequently, development using a concentrated DAB kit, rinsing, counterstaining using 0.1% hematoxylin at room temperature for 3 min, dehydration using ethanol, transparentization using xylol and mounting were performed in sequence. The coverslips were observed under an optical microscope (XDZ-103; Shanghai Tiancheng Medical Flow Technology Co., Ltd., Shanghai, China) with Image Analysis 11.0 software (Beijing Changheng Rongchuang Technology Co., Ltd., Beijing, China).