Fig. 6

tTmod3-N promotes cancer cell invasion by actin remodeling. A Confocal images of A172 cells overexpressing ZsGreen1-tagged tTmod3-N or tTmod3-C (left panel) and co-localization tracer profiles along the line (white arrows) were indicated (right panel). Red, F-actin stained with phalloidine. Scale bar, 20 μm. B Co-immunoprecipitation assays for the interaction of Tmod3 or tTmod3-N with actin in A172 cells with Tmod3-Flag or tTmod3-N-Flag expression. C Lysates of prokaryotic expression of his-Tmod3, his-tTmod3-C and his-tTmod3-N in bacteria were stained with Coomassie brilliant blue R250. Red asterisks mark the targeted proteins. D Actin polymerization stimulated by Tmod3 or truncations of Tmod3 (200 nM). Actin polymerization was measured using Actin Polymerization Biochem Kit (BK003). E Representative fluorescent staining images of F-actin and G-actin in the indicated group of U87-MG and A172 cells. Yellow, F-actin; Red, G-actin. Scale bar, 20 μm. F Statistics of the F-actin/G-actin ratio of the indicated groups of U87-MG and A172 cells. Fluorescence intensity is captured by Image J. Fifty cells were randomly selected from each group for calculation. G Western blotting for F-actin and G-actin in U87-MG and A172 cells following cellular fractionation. The blots are representative of n = 3 independent experiments. H Statistics of the F-actin/G-actin ratio in the indicated groups of U87-MG and A172 cells. I Representative immunofluorescent staining of Cortactin and phalloidin staining of the indicated groups of U87-MG and A172 cells. Arrows indicate the co-localization of Cortactin with F-actin, which represents invadopodia. Scale bar, 40 μm. J Percentage of U87-MG and A172 cells with invadopodia. Five fields were randomly selected in each group. NC = negative control, cells expressing scramble shRNA. KD = knockdown, cells expressing AEP shRNA. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ^****P < 0.0001, n.s. = no significance