Dapi staining solution

2 × 10⁴ cells/well 4T1 cells were inoculated in a 24-well culture plate with cell slides placed at the bottom in advance, so that the cells expand to 60–70% before experiment. The cells on the slides were washed by PBS for three times and then fixed with 500 μl 4% polyformaldehyde at room temperature for 15 min. After that, pre-cooled 0.2% Triton X-100 diluted in PBS was used to permeabilize the cells for 5 min. Then, the cells in each well were blocked with 500 μl goat serum containing 0.3% Triton X-100 at room temperature for an hour. After removal of the goat serum, the cells were covered with the mixed primary antibodies of E-cadherin (CST, Boston, Massachusetts, USA) and vimentin (Abcam, Cambridge, UK) overnight at 4 °C under dark conditions, and the two primary antibodies were coupled with Alexa Fluor® 594 and Alexa Fluor® 488, respectively. Afterwards, cells were washed three times using PBS, stained with DAPI staining solution (Invitrogen, Camarillo, CA, USA) for 10 min, washed, and sealed with BrightMount (Abcam, Cambridge, UK). Cells on the slides were observed by using a fluorescence confocal microscope (Zeiss, Oberkochen, Germany) with images subsequently obtained.

Immunofluorescence was used to determine phosphoryl-NF-κB p65 localization. GH3 cells were fixed with paraformaldehyde (v/v, 1/25) at 37°C for 10 minutes. They were then permeabilized with cold acetone at −20°C for 3 minutes. After a PBS wash (0.1 mM, pH 7.4), cells were saturated with 3% BSA in PBS for 30 minutes, and incubated with the anti-phosphoryl-NF-κB p65 antibody (diluted 1 : 100) at 4°C overnight. After another PBS wash (0.1 mM, pH 7.4), the cells were incubated with the secondary antibody for 30 minutes at room temperature. Coverslips were washed twice with PBS (0.1 mM, pH 7.4), incubated with the goat anti-mouse IgG antibody conjugated with Alexa Fluor 555 (Cell Signaling Technology, Danvers, MA, USA) for 30 minutes in the dark, incubated in 5 μM DAPI staining solution (Invitrogen) for 5 minutes, and then washed in PBS. The fluorescence was monitored using an UltraVIEW VoX confocal system (PerkinElmer, Co., Norwalk, CT, USA).

4T1 cells were inoculated into a 24-well culture plate with pre-placed cell slides. The cells were treated with 4% paraformaldehyde for 15 min and then placed in 0.2% Triton X-100 at 4 °C for 5 min. Non-specific antibodies were blocked with goat serum containing 0.3% Triton X-100 (room temperature, 1 h). The mixed primary antibodies, including E-cadherin (CST, Boston, Massachusetts, USA) and vimentin (Abcam, Cambridge, UK), were placed over the slides and incubated for 12 h at 4 °C in a light-proof environment. Alexa Fluor 594 and Alexa Fluor 488 were coupled to the above two primary antibodies and the nuclei were stained with DAPI staining solution (Invitrogen, Camarillo, CA, USA). The slides were sealed and observed under a fluorescence confocal microscope (Zeiss, Oberkochen, Germany) and photographed.

Cells were rinsed three times in PBS and fixed with cold methanol, washed thoroughly with PBS, incubated with DAPI staining solution (1 µg/mL, Thermo Fisher Scientific, Waltham, MA, USA) for 30 minutes (min) and rinsed three times with PBS, followed by viewing using a fluorescence microscope (Olympus, Tokyo, Japan).
For the immunofluorescent staining, the human glioma cells U87MG and M059J were cultured on the coverslips and treated with CA (25 µM, 50 µM) for 24 h, Subsequently, the cells on the coverslips were fixed with 4% paraformaldehyde (15 min at 4 °C), blocked with 1% BSA (1 h, RT), and incubated with primary antibody, rabbit anti-GFAP antibody (1:200 dilution; Proteintech, IL, USA), or mouse anti TUBB3-antibody (Tuj1, 1:200 dilution; Proteintech, IL, USA) for overnight at 4 °C. The coverslips were then washed 3 times with PBS and stained with a Cy3-conjugated anti-mouse secondary antibody or an Alexa Fluor 488-conjugated anti-rabbit secondary antibody (1:200 dilution; Thermo Fisher Scientific, Waltham, MA, USA). Cell nuclei were counterstained with DAPI (1 µg/mL, Thermo Fisher Scientific, Waltham, MA, USA). Confocal images were taken using a fluorescent microscope (Nikon Eclipse CI, Japan) and fluorescence pictures were photographed using the Nikon DS-U3 system (Japan).