For cell proliferation assay, the equal cell number seeded in the 6-well culture plates were transfected with control siRNA or siRNA for MAP4 knockdown. Cell numbers were counted 96-hrs post-siRNA transfection after detaching the cells from the culture plates. For wound healing, cells were seeded to 6-well plates and transfected by control and MAP4 siRNA for 48 hrs until achieving confluence. Then the cells were starved in serum-free medium for 24 hrs and treated with 10 ng/ml EGF. The cellular layer in each plate was scratched using a plastic pipette tip. The migration of the cells at the edge of the scratch was imaged at 0, 6, 12, 24 and 48 hrs using the Nikon Eclipse TE2000-U microscope (Nikon Instruments Inc) and quantified by ImageJ.
The bottom polycarbonate filter surface of Transwell inserts (8 μm pores; Corning) was coated with 10 μg/ml of LN332 (Kerafast) diluted in PBS for 3 hrs at 37oC. Cells (5×104) 48 hrs post-transfection of control or MAP4 siRNA were suspended in serum-free medium containing 1% BSA and then were plated in the upper insert chamber with or without 10 ng/ml EGF. Cells were allowed to migrate/invade for 16 hrs at 37°C. Cells on the bottom of the filter were then fixed with 4% PFA and stained with 0.1% Crystal Violet. Then the cells were imaged by the Nikon Eclipse TE2000U microscope and quantified by ImageJ.
The bottom polycarbonate filter surface of Transwell inserts (8 μm pores; Corning) was coated with 10 μg/ml of LN332 (Kerafast) diluted in PBS for 3 hrs at 37oC. Cells (5×104) 48 hrs post-transfection of control or MAP4 siRNA were suspended in serum-free medium containing 1% BSA and then were plated in the upper insert chamber with or without 10 ng/ml EGF. Cells were allowed to migrate/invade for 16 hrs at 37°C. Cells on the bottom of the filter were then fixed with 4% PFA and stained with 0.1% Crystal Violet. Then the cells were imaged by the Nikon Eclipse TE2000U microscope and quantified by ImageJ.