Control chambers

Transwell migration and invasion assays were performed with Millicell hanging Biocoat Matrigel and control chambers from BD Biosciences (24-wellinsert, 8-lm pore size) as previously described [15 (link)]. Briefly, 4 × 10⁴ cells in 200 μL serum free medium were loaded into the upper chambers, while lower chambers were filled with 500 μL custom medium. In migration assays, the cells on the underside of the membrane were stained 24 h later. In invasion assays, the cells on the underside of the membrane were stained 48 h later. Then, the cells were counted under a microscope.

Control cells and cells stably expressing ApoF were plated into 96-well plates at a density of 1000 cells/well in DMEM with 10% FBS. At indicated time points, cell proliferation was determined using a cell-counting kit-8 (CCK-8; DOJINDO, Kumamoto, Japan) assay.
Transwell assays were conducted using Millicell hanging BioCoat Matrigel and control chambers from BD Bioscience (24-well insert, 80-µm pore size). Briefly, 25 × 10⁴ cells in 200 µL of serum-free DMEM were loaded into the upper chambers of the transwell device. A total of 0.5 mL DMEM supplemented with 10% FBS was loaded into the lower chamber. After 24 h of treatment, the cells on the underside of the membrane were stained and counted under a microscope in five random high-power fields.