Figure 1.
PTK6 is expressed in pancreatic cancer cell lines and human pancreatic adenocarcinoma tissues at various levels.
A, Endogenous expression of PTK6 in human pancreatic cancer cell lines. PTK6 expression was confirmed in 5 pancreatic cancer cell lines, BXPC3, Capan1, Hs766T, MIAPaCa2, and Panc1 at various levels by Western-blotting. B, Immunohistochemical staning for PTK6 in adjacent normal pancreas and pancreatic cancer tissue. While there was no detectable PTK expression in adjacent normal pancreatic ductal epithelium (upper left), PTK6 was expressed at various levels (from negative through strongly positive) in pancreatic cancers.
Figure 2.
PTK6 gene silencing reduces cell migration and invasion of pancreatic cancer cells.
A, PTK6 gene silencing. Cells were transfected with PTK6-specific siRNA (siRNA-PTK6) or negative control-siRNA (siRNA-NC) for 96 hours. Knockdown of PTK6 expression was comfirmed by Western-blotting. The numbers at the bottom indicate the relative intensity of the bands for PTK6 to corresponding controls (normalized by β-actin). B, C, The effect of PTK6 gene silencing on cell motility (B) and invasion (C). Cells were treated with PTK6-specific siRNA or negative control-siRNA for 48 hours, then subjected to migration or invasion assay using Boyden chambers without or with Matrigel. Gene silencing of PTK6 reduced cellular migration in all 3 pancreatic cancer cell lines (0.59 fold decrease in BXPC3, 0.61 in Panc1, 0.42 in MIAPaCa2, respectively, *p<0.05 by t-test). Similarly, gene silencing of PTK6 reduced cellular invasion in all 3 pancreatic cancer cell lines (0.49-fold decrease in BXPC3, 0.62-fold in Panc1, 0.39-fold in MIAPaCa2, respectively, *p<0.05 by t-test). Each assay was performed in triplicate.
Figure 3.
PTK6 overexpression increases cell migration and invasion of pancreatic cancer cells.
A, Overexpression of PTK6. Cells were transfected with pcDNA3.1-Mock (empty vector) or pcDNA3.1-PTK6. Overexpression of PTK6 in stable trasfectant was confirmed by Western blotting in Panc1 and MIAPaCa2 cells, respectively. The numbers at the bottom indicate the relative intensity of the band for PTK6 to the corresponding controls (normalized by β-actin). B, C, The effect of PTK6 overexpression on cellular migration (B) and invasion (C). Established transfectant cells were evaluated with migration and invasion assays. Forced overexpression of PTK6 increased cellular migration in Panc1 and MiaPaCa2 pancreatic cancer cells (1.6-fold increase for Panc1, and 1.4-fold for MIAPaCa2, respectively, *p<0.05 by t-test). Similarly, forced overexpression of PTK6 increased cellular invasion in these 2 cell lines (1.7-fold increase for Panc1, and 1.6-fold for MiaPaCa2, respectively, *p<0.05 by t-test). Each assay was performed in triplicate.
Figure 4.
Alteration in PTK6 expression affects ERK1/2 activation.
A, Effect of PTK6 gene silencing on total ERK1/2 and activated (phosphorylated) ERK1/2. Gene silencing of PTK6 reduced the level of phosphorylated ERK1/2 in all 3 pancreatic cancer cell lines. The numbers at the bottom indicate the relative band intensity for pERK1/2 to corresponding controls (normalized by total ERK1/2). B, Effect of PTK6 overexpression on total ERK1/2 and activated ERK1/2. Forced PTK6 overexpression induced activation of ERK1/2 in both Panc1 and MiaPaCa2 cells. The numbers at the bottom indicate the relative band intensity for pERK1/2 to corresponding controls (normalized by total ERK1/2).
Figure 5.
The effect of ERK1/2 inhibition on PTK6-induced cellular migration and invasion.
A, Cells were treated with the specific ERK1/2 inhibitor, U0126 at 10 µM for 24 hours. ERK1/2 phosphorylation was inhibited and PTK6-induced activation of ERK1/2 was no longer detected in Panc1 or MIAPaCa2 cells. B, The effect of ERK1/2 inhibition on cellular migration (left) and invasion (right) ERK1/2 inhibition not only reduced basal migration and invasion in Panc1 and MIAPaCa2 cells, but abolished the effect of PTK6-overexpression on migration and invasion. *p<0.05 vs corresponding control with DMSO vehicle (by t-test). **p<0.05 vs PTK6-overexpressed cells with DMSO vehicle (by t-test). Each assay was performed in triplicate.