Transwell migration and invasion assays

Transwell migration and invasion assays were carried out using a chamber containing a polycarbonate filter with a pore size of 8.0 μm (24-well insert; Corning, NY, USA). Polycarbonate filters precoated with Matrigel Matrix (BD Biosciences, San Jose, CA, USA) were used for the invasion assay, and uncoated filters were used for the migration assay. Cells were serum-starved for 8 h. The lower chambers were filled with 800 mL of MEM (SiHa/C-33A) or DMEM (HeLa) supplemented with (concentrations of 2.5–20 mM) or without PLA. Approximately 3–5 × 10⁴ cells were resuspended in 300 μL of serum-free medium and added to the upper chamber. After incubation for 16 h at 37 °C and 5% CO₂, the lower chambers were incubated with calcein-AM (Thermo Fisher Scientific) to stain the cells that invaded through the uncoated membrane or the Matrigel Matrix. The stained cells were counted in 4 randomly selected visual fields using a CCD camera mounted to an inverted microscope running MetaMorph image analysis software (Molecular Devices, Sunnyvale, CA).