*2.3. Determination of Migration and Invasion of Cancer Cells following BPL2 Treatment*

BPL2 strongly inhibited the invasion and migration of lung cancer cells in the various cell lines (A549, H460, and H1299). A concentration-dependent effect was observed in both the cell migration and invasion assays. The migration of all cancer cell lines was reduced to less than 10% following treatment with 20 μg/mL of BPL2. The cell invasion rate of lung cancer cells also decreased to 20% at the same treatment conditions (Figure 4).

**Figure 4.** Effect of BPL2 on migration and invasion of cancer cells. Transwell migration (Upper images) and invasion assays (Bottom images) for A549, H460, and H1299 cell lines were performed using different concentrations of BPL2. Representative graphs are shown with the quantification of the randomly selected fields.

The expression levels of EMT-related genes (N-cadherin, E-cadherin, ZEB1, vimentin, and Twist) were investigated to understand the involvement of BPL2 in the EMT pathway. BPL2 induced the downregulation of ZEB1, Twist, vimentin, and N-cadherin and upregulated E-cadherin expression. Gene expression levels showed similar regulation in all tested cancer cell lines, viz. A549, H460, and H1299 (Figure 5A). The levels of EMT-related proteins (N-cadherin, ZEB1, vimentin, and snai1) showed the same trends as their corresponding gene expressions (Figure 5B).

**Figure 5.** Levels of (**A**) EMT gene and (**B**) protein expression. (**A**) Gene expression in A549, H460, and H1299 cell lines following treatment with BPL2. (**B**) The protein expression in A549 cells following treatment with BPL2. β-actin was used as the control.

The levels of EGFR signaling-related protein expression were determined. EGFR in A549 and H460 cells decreased significantly upon BPL2 treatment. Activation of ERK and AKT, important downstream targets of the EGFR signaling pathway, reduced significantly (Figure 6).

**Figure 6.** Levels of EGFR signaling-related protein expression. Level of proteins in A549 and H460 cell lines following treatment with BPL2 as determined by Western blot analysis. β-actin was used as the control.