Dapi solution

Cells were seeded onto coverslips (Ø 13 mm) coated with 5 µg/cm² rat collagen (ibidi, Gräfelfing Germany) for the fluorescence microscopy experiments. Following the cultivation procedure described above, the cells were washed twice in PBS, fixed for 20 min with 4% paraformaldehyde and washed twice again with PBS. Permeabilization was accomplished by 10 min incubation with 0.1% Triton^® X 100 (Roth, Karlsruhe, Germany) in PBS and another two washing steps. The blocking of unspecific binding sites was achieved by the application of 5% bovine serum albumin in PBS for 1 h. Cells were washed twice again with PBS. A primary antibody dilution in 1% bovine serum albumin in PBS was applied (ab137443, 1:100; Abcam, Cambridge, UK) for 2 h. Subsequently, another double washing step was completed before the cells were incubated for 1 h in the presence of a secondary antibody dilution in 1% BSA in PBS (ab150078, 1:400; Abcam, Cambridge, UK). After washing twice, the cells were counterstained with a 0.25 µM DAPI solution (Abcam, Cambridge, UK). After a final double wash step, the coverslips were mounted in ROTI^® Mount FluorCare mounting media (Roth, Karlsruhe, Germany). A BZ-X810 microscope (Keyence, Osaka, Japan) was used to capture fluorescence images.

Cells were seeded into confocal microscope compatible dishes (Cellvis), which were precoated with Matrigel. After preparation for scanning, cells were fixed with 4% paraformaldehyde at RT for 10 min, permeabilized with 0.1% Triton X-100 (Invitrogen) at RT for 20 min, and then blocked with 3% BSA at RT for 1 h. Cells were incubated with primary antibodies at 4 °C overnight and secondary antibodies at RT for 1 h. Afterward, cells were incubated with DAPI solution (Abcam) at RT for 5 min. Images were obtained by confocal microscope scanning microscopy(TCS SP5 II, Leica Microsytems).

SKBR3 and ZR75 cells were grown on coverslips and stained for immunofluorescence. Briefly, cells were treated with a combination of DA and BMS-202 for 48 h. Cells were washed with PBS and fixed with 4% of formaldehyde, followed by permeabilization using 0.2% of triton X-100. Cells were then washed and blocked with a 10% FBS blocker. Then, they were incubated with the primary antibody of E-cadherin and β-catenin (Abcam, Cambridge, MA, USA) overnight in a humidified chamber. On the next day, cells were washed and incubated with the corresponding secondary antibody in the dark. Afterward, DAPI staining with 300 ng/ml of DAPI solution (Abcam, Cambridge, MA, USA) was performed in the dark, then cells were mounted using Jelly mount water-based mounting media (DDk Italia, #04-108), and fluorescence was visualized by fluorescence microscope. Untreated cells served as a control.

Immunofluorescent antibody staining was used to identify CD133, nestin, and FRAT1 expression in GSCs. Cultured GSCs were placed on glass slides coated with 40 g/l polylysine solution and post-fixed in 4% paraformaldehyde for 15 to 20 min, followed by washing three times in 0.01 mol/l PBS for 5 min each. After blocking non-specific antigen on the cell surface using 1% BSA at 37°C for 1 h, cells were incubated with 1:300 PBS-diluted primary antibody at 4°C for 8 h and 37°C for 1 h. Then, cells were incubated with 1:200 PBS-diluted fluorescent secondary antibody in the dark at 37°C for 40 min. Cell nuclei were eventually stained with DAPI solution (Abcam). After washing the stained cells with PBS, cytomorphology was observed with a fluorescent microscope and photographed with a digital camera. Primary antibodies used included rabbit anti-human CD133 monoclonal antibody (Abcam), rabbit anti-human nestin monoclonal antibody (Abcam), and mouse anti-human FRAT1 monoclonal antibody (Abcam). Secondary antibody used included Alexa Fluor 488 (goat anti-rabbit fluorescent antibody, Cell signaling technology, Danvers, MA) and Alexa Fluor 555 (goat anti-mouse fluorescent antibody, Cell signaling technology).