Figure 4. EF24 inhibits CCA tumor metastasis.

(A) Migration (Left-panel) and invasion (Right-panel) assays of the indicated CCA cell lines. Cells that migrated and invaded to the bottom of the chamber were counted in three fields under × 20 magnifications. (B) Single and merged images were taken to show immunofluorescence staining of N-cadherin (green) and vimentin (red) accompanied by the cell nucleus (blue) stained by DAPI. (C) 24 hours after treatment, immunoblotting showed EF24 (2 μM) increased the expression of E-cadherin and decreased the expression of N-cadherin and vimentin in CCA cells. (D) EF24 inhibited HuCCT-1 cells stress fiber and focal adhesion formation. HuCCT-1 cells were treated with DMSO or EF24. Cells were stained with rhodamine-phalloidin and vinculin antibody (Green = vinculin, red = F-actin). (E) The multiple tumor masses formed by the HuCCT-1 cells in the control group were much less than that by HuCCT-1 cells in the EF24 treated group. All data were the means ± SD of three separate experiments. *P < 0.05, **P < 0.01, ***P < 0.001.