Fig 2: Changes in angiogenic potential following ILK knockdown in Gem-R (MIA PaCa-2, AsPC-1) PaCa cell lines. (A) IL-8 mRNA expression after transfection with ILK siRNA was assessed using reverse transcription-quantitative polymerase chain reaction. (B) IL-8 secretion following transfection with ILK siRNA was measured using ELISA. (C) Angiogenic potential after transfection with ILK siRNA was assessed using Matrigel-based tube formation assays. Human endothelial cells (EAhy.926 cells) were seeded in Matrigel at 1.2×105 cells/well and cultured for 16 h in conditioned medium. The parental cell lines for Gem-R PaCa cell lines were used as controls. The number of endotubes was counted, and comparisons among groups were performed using one-way analysis of variance with post hoc Bonferroni tests. *P<0.05. ILK, integrin-linked kinase; Gem-R, Gem-resistant; siRNA, small interfering RNA; ncILK, negative control ILK; siILK, ILK siRNA.

Fig 3: Changes in angiogenic potential following ILK knockdown in pancreatic cancer cell lines. (A) IL-8 mRNA expression after transfection with ILK siRNA was assessed using reverse transcription-quantitative polymerase chain reaction. (B) IL-8 protein secretion following transfection with ILK siRNA was assessed using ELISA. (C and D) Angiogenic potential after transfection with ILK siRNA was determined using tube formation assays. EA.hy926 cells were seeded at 1.2×105 cells/well and cultured with conditioned medium for 16 h. The control media was 1% RPMI-1640 containing FBS. The number of endotubes was counted, and differences among groups were evaluated using one-way analysis of variance with post hoc Bonferroni tests. *P<0.05. ILK, integrin-linked kinase; siRNA, small interfering RNA; ncILK, negative control ILK; siILK, ILK siRNA.

Fig 4: Comparison of ILK expression in human pancreatic ductal epithelial cells and PaCa cells. (A) ILK mRNA expression in HPDE cells (H6C7) and PaCa cells (AsPC-1, BxPC-3, Capan-2, MIA PaCa-2, PANC-1, SW1990) was assessed using reverse transcription-quantitative polymerase chain reaction. The mRNA expression status for each sample was normalized to the expression of GAPDH. (B) ILK protein expression levels in HPDE cells (H6C7) and PaCa cells (AsPC-1, BxPC-3, Capan-2, MIA PaCa-2, PANC-1, SW1990) were evaluated using western blotting. (C) The relative expression of ILK compared with GAPDH was assessed. Significant differences in ILK expression between HPDE (H6C7) and PaCa cells were assessed using one-way analysis of variance. *P<0.05. PaCa, pancreatic cancer; ILK, integrin-linked kinase.

Fig 5: Changes in invasive potential following ILK knockdown in pancreatic cancer cell lines. (A) ILK mRNA expression following transfection with ILK siRNA was assessed using reverse transcription-quantitative polymerase chain reaction. (B) ILK protein expression after transfection with ILK siRNA was assessed using western blotting. (C and D) PaCa cells (1.0×105) were seeded into Matrigel-precoated Transwell chambers, and the number of invasive cells was determined after 24 h. Cells that passed through the Matrigel and invaded into the bottom of the upper chamber were fixed and stained using Diff-Quik staining. A comparison of the mean number of invasive cells in nine random microscopic fields of view is presented. Comparisons among groups were performed using one-way analysis of variance with post hoc Bonferroni tests. *P<0.05. ILK, integrin-linked kinase; siRNA, small interfering RNA; ncILK, negative control ILK; siILK, ILK siRNA.