Fig. 7

Association of CCDC88A and AMPK1 with cell migration and invasion. a. Effects of suppression of CCDC88A on the expression of selected phosphoproteins in S2-013 cells. Cell extracts obtained from fibronectin-stimulated scrambled control-siRNA transfected S2-013 cells or CCDC88A-siRNA transfected S2-013 cells were probed on human phosphoprotein arrays. b. Confocal immunofluorescence microscopic images of S2-013 cells that were cultured on fibronectin and were then labeled with anti-AMPK1 antibody (green), anti-CCDC88A antibody (red) and phalloidin (violet; actin filaments). Arrows, AMPK1 localized in cell protrusions. Blue, nuclear DAPI staining. Bar, 10 μm. c. Western blot analysis of AMPK1 following transient transfection of S2-013 and PANC-1 cells with a single mixture containing four different siRNA oligonucleotides targeting AMPK1 (siAMPK1) or negative scrambled control (Scr). Western blotting was performed using an anti-AMPK1 antibody. d. Confocal immunofluorescence microscopic images. A myc-tagged CCDC88A-rescue construct was transfected into S2-013 and PANC-1 cells that had been transfected with both CCDC88A-siRNA and AMPK1-siRNA. 48 h later, the cells were incubated on fibronectin. Cells were stained with anti-myc antibody (violet), anti-AMPK1 antibody (green), and phalloidin (red). Blue, DAPI staining. Bars, 10 μm. e. siRNA oligonucleotides targeting AMPK1 or Scr were transiently transfected into S2-013 and PANC-1 cells. After 48 h, migration and two-chamber invasion assays were performed. Migrating cells in four fields per group were scored (lower panel). Data are representative of three independent experiments. Columns, mean; bars, SD. *p < 0.008 compared with Scr-transfected control (Student’s t-test). f, g. A myc-tagged CCDC88A-rescue construct was transfected into S2-013 (f) and PANC-1 (g) cells that had been transfected with CCDC88A-siRNA and AMPK1-siRNA; 48 h later, migration and two-chamber invasion assays were performed. Migrating cells in four fields per group were counted. Data are derived from three independent experiments. Columns, mean; bars, SD. *p < 0.005 compared with corresponding CCDC88A-siRNA and AMPK1-siRNA transfected cells that were transfected with mock vector (Student’s t-test)