Crystal violet

Transwell chambers (CORNING, Corning, NY) were chosen to explore the impact of miR-203a-3p on cell migration and invasion. After transfection, 1×10⁵ cells were resuspended in 200 µL DMEM and then transplanted into upper chambers in 24-well plates. Prior to seed cells, the upper surface of chambers was pre-coated using 50 µL Matrigel before the invasion assay. After transfection 24 h, cells that did not pass through the membrane were cautiously scrubbed by cotton swabs. And the cells transferred to the basement of the membranes were fixed with 4% PFA, and dyed with 0.1% crystal violet (Abcam, Cambridge, UK) for 40 min. The number of migrating and invading cells were photographed and counted 5 randomly selected fields by using a phase contrast microscope (×200) (Zeiss, Oberkochen, Germany).
About the wound healing experiment, seeded 1×10⁵ transected cells per well into 24-well plates, then subcultured to form a monolayer. When the cell density reached over 90%, the monolayer was scratched by using a 100 µL pipette. Then washed the cells twice with DMEM and appended fresh serum-free medium to further incubation. Photos were taken randomly with microscopy (Zeiss, Oberkochen, Germany) at 0 h, 24 h and 48 h. Wound healing rate (%) = (x h Scratch area − 0 h Scratch area)/0 h Scratch area ×100.

Cell proliferation was detected by colony formation assay. In detail, THP-1 cells (1 × 10³ cell/per well) were added to a 6-well cell culture plate. After 7–10 days, the cells were fixed at 10–15 min with 4% formaldehyde, and the cells were dyed in the crystal violet (Abcam, Cambridge, UK) for 15 min at room temperature, and finally there were 5 cell fields were selected for counting.

U251 and LN229 cells (6 × 10⁴) were suspended in 100 μL serum-free medium. They were then seeded into the upper chamber, and 20% FBS was added to the lower chamber to induce cell invasion through the membrane. Matrigel (1:6 dilution; BD Biosciences) was then added to the upper chamber. After 24 h, cells that invaded across the Transwell membrane and were stained with crystal violet (Abcam, Cambridge, England) that invaded across the Transwell membrane were counted under an optical microscope (Leica, Wetzlar, Germany).