Figure 4.

CPNE8 acted on the Focal adhesion pathway to promote GC metastasis. (A) KEGG functional enrichment analysis revealed a significant correlation between CPNE8 expression and Focal adhesion pathway in AGS-sh-CPNE8 cells compared to the control cells. (B) There are relative changes of FAK (Gene Symbol as PTK2) and ERK mRNA levels in GC cells with knockdown or overexpression of CPNE8. (C) Western blot analysis of FAK, p-FAK, total Erk1/2, and p-ERK protein expression in GC cells with knockdown or overexpression of CPNE8. Relative protein expression (interested protein/GAPDH) was also quantified in GC cells. (D) EDU staining assay showed the proliferating cells with or without diminishing FAK expression, such as GSK2256098 treatment and knockdown of FAK. Photos were captured using a fluorescence microscope (Leica-Microsystems). DNA (blue) was stained with Hoechst. Cyan cells showed EDU/Hoechst-positive cells. The column diagram represented the proliferation rates in various GC cells. Data are presented as mean ± SD for at least three independent experiments. (E) Wound-healing assays revealed that both GSK2256098 and si-FAK could quantitatively reduce the CPNE8-induced migration in BGC823 cells using Image J software. (F) Transwell assays confirmed that GSK2256098 or knocking down FAK could inhibit migration and invasiveness of GC cells with overexpression of CPNE8. (G) The representative images showed the adhesion ability of GC cells with knockdown or overexpression of CPNE8 compared to the control cells and the attenuation of BGC823-oe-CPNE8 with GSK2256098 or knockdown of FAK. The number of GC cells shown in the bar graph that adhered to the plates coated after 2h of incubation was quantified using Image J software.