Figure 5. Involvement of PEA3 in B16F10 cells migration and invasion.

(A) Elevated expression of PEA3 protein in nuclear extracts from B16F10 cells. PEA3 protein of each cell line nuclear extract was determined by western blotting using the anti-PEA3 antibody. Knockdown of PEA3 mRNA in B16F10 cells led to a reduction of FAK mRNA (B) or FAK protein (C). The B16F10 cells were transfected with 2 µg psi-PEA3 or mock plasmid for 48 h, RT-PCR analysis was performed to measure the PEA3 and FAK mRNAs (B), western blot analysis was performed to determine the PEA3,FAK and phosphorylated FAK (Y397 p-FAK) protein levels in the whole lysates of the B16F10 cells (C-1), tubulin or actin was used as a control. (C-2) Densitometric quantification analysis of western blot results in C-1. Densitometric units were normalized to tubulin and then divided by mock group results. (n = 3; ***, p<0.01) (D-E) Knockdown of PEA3 suppressed B16F10 cell invasiveness. After transfected B16F10 cells with mock or psi-PEA3 for 24 h, cells were collected and re-suspended in culture medium at a density of 1 × 10⁶ cells/ml. 100 µl of the cell suspension was plated into the upper wells of Transwell inserts containing 8 µm pore polycarbonate membranes pre-coated with fibronectin (10 µg/ml) on the under surface. The cells were allowed to migrate for 24 h at 37°C, then cells on upper wells were removed gently and those migrated to the under surface were fixed and stained. Representative membranes stained with crystal violet are shown (D-1), the arrows indicated the migrant cells. Scale bar = 200 µm. Quantitative analysis of the number of the cells migrated to the down side of the membrane (D-2). Data are mean ±SEM of three independent experiments. ***P<0.01, vs. mock control. (E) Knockdown of PEA3 in B16F10 cells suppressed the tumor cells migration. The B16F10 cells were transfected with si-PEA3 or mock vector. Then wound-healing scratch motility assays were performed in the fibronectin-coated plates in the presence of serum. Cell migration was assessed at 0 and 48 h. Representative images are shown (E1). Migration of cells into the wound was quantified (E-2). (n = 3; ***, p<0.01).