Transwell 24 insert plates

For the migration assays,^{61 (link)} 2.0 × 10⁴ CXCR4⁻ or CXCR4⁺ ASCs were resuspended in 100 μL of α-MEM medium without FBS and were placed in the upper well of 8-μm pore-size Transwell 24-insert plates (Corning, NY, USA). The migration assay was performed by adding to the bottom well 600 μL of α-MEM medium supplemented with 10% FBS or CXCL12 (50 ng/ml). After 4 h, cells on the bottom of the inserts were fixed with 100% methanol for 30 min and stained with 0.5% crystal violet (Sigma-Aldrich). Then cells that invaded into the lower surface were taken picture by using an 100× microscope (Leica). The stained chambers were eluted with 33% acetic acid solution and quantified the eluent by absorbance at 570 nm.

After transfection with eIF5A2 siRNA, Twist 1 siRNA, or Twist 2 siRNA (100 nM) for 8 h or treatment with GC7 or NAC for 24 h, cells were seeded at 2 × 10⁵ cells/well in the upper chamber of 8-μm pore-size Transwell 24-insert plates (Corning, NY, USA) with appropriate medium without FBS. The upper chambers had been coated previously with 30 μg Matrigel (BD Biosciences, San Jose, CA, USA) and the lower chamber contained the appropriate medium with 10% FBS. After 48 h, cells on the upper side of the filters were cleansed with cotton swabs, and those on the bottom of the inserts were fixed in 4% paraformaldehyde for 30 min and stained with 0.05% crystal violet. Cells that had invaded to the lower surface were counted in at least 10 fields using an inverted phase-contrast microscope (Olympus; magnification, 20 ×) and photographed. Each experiment was repeated at least three times.

Cells were seeded at 2 × 10⁵ cells/well in the upper chamber of 8-μm pore-size Transwell 24-insert plates (Corning, NY, USA) with appropriate medium without FBS. The upper chambers were coated previously with 30 μg Matrigel (BD Biosciences, San Jose, CA, USA for Transwell-Matrigel invasion assay only), and the lower chamber contained appropriate medium with 10% FBS. After 48 h, cells on the bottom of the inserts were fixed in 4% paraformaldehyde and stained with 0.05% crystal violet. Then cells that invaded into the lower surface were counted in at least 10 fields using an 200× microscope (Olympus). Each experiment was repeated at least three times.