Figure 5
From: The drebrin/EB3 pathway drives invasive activity in prostate cancer
Evidence for a role for drebrin/EB3 interactions in PC-3 cell invasion. (a) Immunoblots of a normal human prostate epithelial cell line (PNT2-C2) and human prostate cancer cell lines probed with antibodies against EB1, EB3 and, as a loading control, GAPDH. (b) Immunoblots of PC-3 cells expressing CFP, CFP-EB1M, CFP-EB3M, CFP-EB1 or CFP-EB3 probed with an antibody to GFP that cross-reacts with CFP. Expression levels of related constructs are similar. (c) Effects of transfection of PC-3 cells with CFP, CFP-EB1 CFP-EB3, CFP-EB1M or CFP-EB3M on cell invasion in a 3D invasion assay in the presence of CXCL12 chemotactic gradients. After 48 h, cells on the lower surface of the insert membrane were labelled with an antibody to GFP and counted using confocal fluorescence microscopy. Error bars are mean±s.e.m. from four independent experiments each of two replicates per condition. Significant difference: ***P<0.001. NS, not significant. (d) Immunofluorescence micrographs of PC-3 cells labelled with a GFP antibody that cross-reacts with CFP, and with phalloidin to label F-actin. Cells were transfected with CFP-EB1M or CFP-EB3M, embedded in Matrigel and exposed to a CXCL12 gradient. PC-3 cells expressing CFP-EB3M are rounded and produce short filopodia (arrowheads) but no pseudopods. In contrast, PC-3 cells expressing CFP-EB1M become polarized and extend pseudopods bearing long, tapered filopodia (arrows) and short, uniform filopodia (arrowheads).
