Phase contrast microscopy

Transwell cell culture inserts (pore size 8 μm; Corning Co-Star Corp., Cambridge, MA, USA) were placed in RPMI 1640 medium supplemented with 15% foetal bovine serum in the lower compartment. Podocytes with different pretreatments were harvested with trypsin and resuspended in serum-free medium. The upper chambers were seeded with 1 × 10⁵ cells/ml, which were allowed to attach at 37°C for 12 h. Non-migratory cells were then removed from the upper surface of the chambers, and migrated cells on the lower membrane surface were fixed with 4% paraformaldehyde and stained with haematoxylin. The number of migrated cells was counted in 3 separate fields per membrane at 200× using phase-contrast microscopy (Leica Microsystems, GmbH). The data are presented as the mean ± S.D.

Transwell cell‐culture inserts (pore size 8 μm; Corning Costar Corp., Cambridge, MA, USA) were rinsed once with PBS and placed in RPMI1640 with 10% foetal bovine serum in the lower compartment. The heights of the medium in the upper and lower compartments were maintained at similar levels; thus, bulk flow was not because of a hydrostatic pressure gradient. Podocytes pre‐treated with different conditions were harvested with trypsin and re‐suspended in serum‐free RPMI1640 medium. The upper chambers were seeded with 1 × 10⁴/ml cells, which were then allowed to attach at 37°C for 6 or 12 hrs, respectively. Next, non‐migratory cells were removed from the upper surface of the membrane, and migrated cells were fixed with 4% paraformaldehyde and stained with haematoxylin. The number of migrated cells in the centre of a membrane (one field) was counted using phase contrast microscopy (Leica Microsystems GmbH). The data presented denote the mean ± S.D. of 6 independent experiments.

Matrigel matrix (Becton-Dickson) was liquefied overnight on ice at 4°C. 50 µl of the matrix was aliquoted in 96-well plates and incubated at 37°C for 30–60 min prior to plating 5×10⁴ cells per well. Cells were cultured for 24 hrs and tube formation monitored by inspection by phase contrast microscopy (Leica Microsystems). At the end of the 24 hr incubation, cells were labeled with 1 µM Calcein AM (Trevigen) and cell viability assessed using a fluorescent inverted microscope with a 485 nm excitation filter.

For invasion assay, breast cancer cells were loaded in Transwells with 8 μm pore size filter inserts (Corning Glass, Seoul, Korea) that were precoated with 10 mg/mL growth factor-reduced matrigel (BD Biosciences, Seoul, Korea) on the upper side of the chamber with the lower well filled with 0.8 ml of growth medium. After incubation for 48 h at 37 °C, and For migration assay, we used Transwells with inserts that contained the same type of membrane but without the matrigel coating. After incubation for 24 h at 37 °C. Non-invaded cells on the upper surface of the filter were removed with a cotton swab and migrated cells on the lower surface of the filter were fixed and stained with the Diff-Quick kit (Fisher, Pittsburgh, PA, USA) and photographed (magnification ×20). Invasiveness and motility were determined by counting cells in four microscopic fields per well, and the extent of invasion was expressed as an average number of cells per microscopic field. Cells were imaged by phase-contrast microscopy (Leica Microsystems, Bannockburn, IL). All experiments were repeated three times.

For the invasion assay, the cells (2.5 × 10⁴ cells in serum-free medium) were seeded in the upper well of the Transwell chamber (8-mm pore size; Corning Glass, Corning, New York, NY, USA) that was precoated with 10 mg/mL growth factor-reduced Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). The lower well was filled with 0.8 mL of growth medium containing 10% fetal bovine serum as a chemoattractant. After incubation for 48 h at 37 °C, non-invaded cells on the upper surface of the filter were removed with a cotton swab, and migrated cells on the lower surface of the filter were fixed and stained with a Diff-Quick kit (Fisher, Waltham, MA, USA). Cells were imaged by phase contrast microscopy (Leica Microsystems, Wetzlar, Germany; magnification 10×). Invasiveness is determined by counting cells in five microscopic fields per well, and the extent of invasion is expressed as an average number of cells per microscopic field.