Figure 1

ILK expression is necessary for cell growth and NF-κB activation. (A) Representative fluorescence-based immunohistochemical staining shows the coexpression of ILK (green) and Ki-67 (red) in human gastric tumors and AGS-derived nodules in BALB/c mice. DAPI (blue) was used for nuclear counterstaining. ILK⁺Ki-67⁺ cells in the immunofluorescently stained tissue sections are presented as dot-plots of a FACS-like analysis by using TissueQuest software. The cells were calculated as the percentage and the cell number of the total cells (DAPI⁺ cells) per field. (B) Western blot of ILK expression in cells transfected with shRNAs targeting ILK (shILK) or a control luciferase (shLuc). β-actin was used as an internal control. WST-8-based assay shows the inhibition of cell growth in ILK-silenced cells. (C) The dose-dependent effect of the ILK inhibitor T315 on the inhibition of AGS cell growth. (D) Representative fluorescence-based immunohistochemical staining shows the coexpression of ILK (green) and phosphorylated NF-κB Ser536 (red) in human gastric tumors and AGS-derived nodules in mice. DAPI (blue) was used for nuclear counterstaining. The dot-plots show the coexpression of ILK and NF-κB Ser536. (E) Logistic regression analysis showed a correlation between ILK, phosphorylated NF-κB Ser536, and Ki-67 in 93 gastric cancer specimens tested. R-squared (R²) and P values are shown. (F) EMSA demonstrating NF-κB activation. (G) Luciferase reporter assay shows the activation ratio of NF-κB to control Renilla luciferase in cells treated with the NF-κB inhibitor CAPE (25 μg/mL). (H) Growth of 25 μg/mL CAPE-treated AGS cells. (I) NF-κB activation in shLuc- or shILK-transfected cells. (J) NF-κB activation after 6 h of T315 treatment in AGS cells. For cell growth, colony formation, and luciferase activity, data are mean ± SD from three independent experiments. *P <0.05, **P <0.01, and ***P <0.001 compared with Day 0 or relative control. #P <0.05, ##P <0.01, and ###P <0.001 compared with shLuc.