Ar 0211

The NIH3T3 cells stably expressed empty vector or wild-type SS18-SSX1, and its mutants were plated in six-well plates at 1.0 × 10³ cells per well in growth medium supplemented with 10% FBS and incubated at 37 °C for 3 weeks. The culture medium was changed every 3–5 days. After 3 weeks, cells were fixed in 4% paraformaldehyde (Dingguo Biotechnology, AR-0211) for 30 min at room temperature and stained with 0.1% crystal violet (Sigma-Aldrich, V5265) for 30 min at room temperature, then cells were washed with water and finally took a picture after air-dried. These experiments were conducted in triplicates.

Cell migration and invasion assays were carried out using a 24-well transwell chamber system (Corning, 3422). A transwell apparatus was separated into upper and lower compartments by polycarbonate filters (8-μm pores). For migration assays, the NIH3T3 cells stably expressed empty vector or wild-type SS18-SSX1, and its mutants (4.0×10⁴) were suspended in 200 μL of growth medium in the upper chamber. For invasion assays, the polycarbonate filters were coated with 300 μg/mL matrigel (Corning, 356234) before the cell seeding. The lower chamber contained a 750 μL growth medium supplemented with 10% FBS. After 24 h incubation at 37 °C in a 5% CO₂ incubator, cells on the upper filter surface were removed by wiping with a cotton swab. Filters were then fixed in 4% paraformaldehyde (Dingguo Biotechnology, AR-0211) and stained with 0.1% crystal violet (Sigma-Aldrich, V5265). All cells that migrated to the lower filter surface were counted under a microscope at ×200 magnification. Each assay was performed in triplicates.