Crystal violet

Cell invasion was analyzed using Transwell chambers pre-coated with 20% Matrigel (Corning, NY, United States) for 20 h at 37°C. Briefly, 5 × 10⁴ cells in serum-free MEM were added to the upper chambers of Transwell inserts, while MEM plus 10% FBS (chemoattractant) was added to the lower chambers. Following incubation for 20 h at 37°C, non-invaded cells were removed, and invading cells in the lower chambers were fixed in absolute ethanol for 10 min and stained in 0.5% crystal violet (Servicebio, China) for 10 min at room temperature. Invaded cells were counted in one randomly selected field from each chamber using an inverted light microscope (magnification ×100; Olympus).

After being infected for 24 h, infected cells were harvested and placed in the upper chambers of transwell inserts (Corning, USA), with a non-serum medium. In the lower chambers, 900 mL of medium containing 10% FBS was added, and the system was maintained for 48 hours. The cells were then fixed with methanol and stained with 0.5% crystal violet (Servicebio). The number of migrated cells in the lower chambers was quantified to reveal the cell invasion ability.

For colony-formation assays, six-well plates were seeded with 500 cells per well, and the cells were cultured for approximately two weeks. Four percent paraformaldehyde (Biosharp, Anhui, China) was used to fix the colonies, and 0.1% crystal violet (G1014, Servicebio, Wuhan) was used for staining. Then, representative colonies were captured and quantified.

For the colony formation assay, EJ and UMUC3 cells stably transfected with plasmids were seeded in 6-wells plates at density of 600 cells per well and cultured for 2 weeks. The cell colonies were fixed with 4% polyformaldehyde for 30 min and stained with 0.1% crystal violet (Servicebio) for another 20 min at room temperature. Cell colonies with more than 50 cells were counted.

For migration assays, human transfected MNNG-HOS and U2OS cells resuspending in serum-free media were seeded at 2 × 10⁴ cells per well into 24-transwell inserts. The inserts were then placed into compete growth media, and cell migration was allowed for 24 h. The cells that had migrated to the basal side of the membrane were quantified under an inverted microscope at 100× magnification after crystal violet (0.1%, Servicebio) staining.

Appropriate numbers of cells (1000, 1200, 1400, 1600, 1800 or 2000/well) were seeded into 6-well plates depending on the different radiation doses and exposed to 0, 2, 4, 6, 8 or 10 Gy radiation respectively. Cells were further allowed to grow for 14 days to form clusters, followed by 4% paraformaldehyde fixation and crystal violet (G1014, Servicebio, Wuhan, China) staining. Colonies comprising more than 50 cells were counted. The calculation formulae for survival fraction (SF): SF = (number of colonies counted / number of colonies seeded) test / (number of colonies counted / number of colonies seeded) control, where “test” denotes the test condition (some radiation dose) and “control” denotes identical cells without radiation.