Biocoat invasion chamber

HUVECs (2 × 10⁴) were plated in the upper chamber of BioCoatTM Invasion Chambers (BD, Bedford, MA) and incubated with conditional medium collected from ESCC cells infected with shR-PLCE1, U73122, and Bay11–7082 at 37 °C for 22 h, followed by removal of cells inside the upper chamber with cotton swabs. Migratory cells on the lower membrane surface were fixed in 1% paraformaldehyde, stained with hematoxylin, and counted (ten random × 200 fields per well). Cell counts are expressed as the mean number of cells per field of view. Three independent experiments were performed, and data are presented as mean ± standard deviation (SD).

Cells (1 × 10⁴) were plated on the top side of polycarbonate Transwell filters coated with Matrigel in the upper chamber of BioCoat^TM Invasion Chambers (BD Biosciences) and incubated at 37 °C for 24 h, followed by removal of cells inside the upper chamber using cotton swabs. Migrated cells on the lower membrane surface were fixed in 1% paraformaldehyde, stained with hematoxylin, and counted (ten random fields per well, ×100 magnification).

Cells (2 × 10⁴) in serum free medium were added on the top side of the polycarbonate Transwell filter without (for Transwell migration assay) or with Matrigel coating (for Transwell matrix penetration assay) in the upper chamber of the BioCoat^TM Invasion Chambers (BD) and incubated at 37°C for 20 hrs (Silencing CLC-3 by ShCLC-3 adenovirus did not significantly reduce the glioma cell numbers at 20 hrs as confirmed by prior MTT assay, Supplementary Figure 1), followed by removal of cells inside the upper chamber with cotton swabs. Migrated and invaded cells on the membrane bottom surface were fixed with methanol, stained with 1% crystal violet and counted using a light microscope in 5 random visual fields at the magnification of 100X.

HUVECs (2 × 10⁴) were plated in the upper chamber of the BioCoat^TM Invasion Chambers (BD, Bedford, MA) and incubated with the conditional medium collected from HCC cells infected with Vector, AGK or Scramble, AGK-RNAi at 37°C for 22 hours, followed by removal of cells inside the upper chamber with cotton swabs. Migratory cells on the lower membrane surface were fixed in 1% paraformaldehyde, stained with hematoxylin, and counted (Ten random 200 × fields per well). Cell counts are expressed as the mean number of cells per field of view. Three independent experiments were performed and the data are presented as mean ± standard deviation (SD).

For invasion assay, U2OS and MG63 cells transfected with TEM8 siRNA or Scrambled siRNA were trypsinized and resuspended to a density of 1 × 10⁵/ml in serum-free medium. A 200-ul cell suspension was plated into the top side of polycarbonate Transwell filter coated with 30 mg/cm² of Matrigel in the upper chamber of the BioCoatTM Invasion Chambers (BD, Bedford, MA). Each well was sustained 400 ul DMEM medium or RPMI 1640 medium respectively. After incubation at 37 °C for 36 h, cells on the upper chamber were removed by wiping gently with cotton swabs. Cells on the lower membrane surface were fixed with 1% paraformaldehyde for 20 minutes, stained with 0.3% Crystal Violet solution for 20 minutes, and then counted (five high-power fields per chamber). The migration assay was performed by the same procesure as the invasion assay except that no matrigel was added to the transwell membrane. Both experiments had three independent replicates and the data were presented as mean ± standard deviation (SD).

SW480 cells were transfected with lenti-virus expressing Gankyrin (Lenti-Gankyrin) or empty lentiviral vector (Lenti-vector). SW480-vector cells and SW480-Gankyrin cells were added to the upper chamber of the polycarbonate Transwell filter (pre-coated with Matrigel) in the BioCoatTM Invasion Chambers (BD Biosciences, San Jose, CA) respectively. RPMI1600 medium with 10% FBS was added to the lower chamber. The cells were allowed to migrate for 12 h at 37°C. After removing cells on the upper side of the transwell, the invading cells on the underside were fixed and stained with Giemsa solution. The stained invasive cells were photographed under a microscope. Three independent experiments were performed and the data are presented as the mean ± standard deviation (SD).