Axio vert a1

The treated 4T1 and MDA-MB-231 cells were seeded into a 6-well plate and incubated at 37°C overnight, after which the cells (85%–95% confluence) were carefully scraped out utilizing a 200 μL pipette tip. Each well was washed with PBS twice to smooth the edges of the scratches and remove the floating cells. Subsequently, the medium containing the tested drugs was added to the wells. The imaging of the plate was conducted at the same location using an inverted optical microscope (Zeiss Axio vert A1, Oberkochen, Germany). The gap was measured at 0 h (t1), and 24 h (t2), and the relative mobility (%) was derived based on the formula: (t1-t2)/t1×100%.
In terms of transwell migration assay, 6×10⁴ MDA-MB-231 cells or 5×10⁵ 4T1 cells in 200 μL of serum-free or drug-containing medium were introduced into the upper chamber of a transwell insert, and 600 μL of the serum-containing medium was added into the lower chamber of transwell plates (3422, Corning, USA). After 24 h incubation, the cells were fixed with 600 μL methanol for 30 min. Subsequently, the cells were stained with 600 μL 0.1% crystal violet (LEAGENE, Beijing) for 30 min. The micrographs were imaged at 10× magnification under a microscope (Zeiss Axio vert A1, Oberkochen, Germany). The stained cells were quantified from three random fields.

For calcification assessment, MC3T3-E1 cells were cultured (1×10⁴ cells/well in 6-well plates) under the same culture conditions as detailed above for 14 days. Alizarin Red staining of matrix mineralization was performed. Cells were fixed in 4% paraformaldehyde for 10 min at room temperature and stained with 0.1% Alizarin Red (Sigma-Aldrich; Merck KGaA)/Tris-HCl (pH 8.3) for 30 min at 37°C, air-dried and photographed under a light microscope at ×100 magnification (4 images/well; ZEISS Axio VertA1, Zeiss AG, Oberkochen, Germany). The calcified nodules were quantized by integrated optical density using Image Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).