Figure 6

EB1 and EB3 contribute to PC-3 cell invasion. (a) Immunofluorescence images of a human prostate cancer PC-3 cell grown on a glass coverslip and labelled with antibodies against EB1, EB3 and tyrosinated α-tubulin (tyr-tubulin), to identify dynamic microtubules. EB1 localizes to the tip of dynamic microtubules (arrows) and has a shorter and more distal localization than EB3 (arrowheads). (b) Fluorescence intensity line plots of EB1 (green), EB3 (red) and tyr-tubulin (blue) in PC-3 cells. (c) Quantitative analysis of the length distribution of EB1 and EB3 on dynamic microtubules measured from fluorescent intensity line plots in PC-3 cells transfected with either control siRNA (Con siRNA) or EB1 siRNA 1 and 2 or EB3 siRNA 1 and 2 and probed with antibodies against EB1 and EB3. Error bars are mean±s.e.m. from three independent experiments each of at least thirty measurements. Significant difference: ***P<0.001. (d) Immunoblots of PC-3 cells transfected with control siRNA (Con siRNA) or EB1 siRNA 1 or 2 or EB3 siRNA 1 or 2 and probed with antibodies against EB1, EB3 and GAPDH. EB1 siRNA specifically knocks down EB1 levels by >95% compared to control and EB3 siRNA specifically knocks down EB3 levels by >55% compared to control. (e) Immunofluorescence images of human prostate cancer PC-3 cells grown on glass coverslips and labelled with antibodies against EB1 and EB3. Cells were transfected with control siRNA or with EB1 siRNA 1 and 2 or EB3 siRNA 1 and 2. In control cells (Con siRNA), EB1 localizes to the tip of dynamic microtubules and has a shorter and more distal localization than EB3. In cells lacking EB1 (EB1 siRNA), the distribution of EB3 along microtubules appears normal whereas in cells lacking EB3 (EB3 siRNA), the distribution of EB1 along the microtubule increases. (f) Effects of EB1 and EB3 knockdown with siRNA on PC-3 cell invasion in a 3D invasion assay in the presence of CXCL12 chemotactic gradients. Cells were transfected with control siRNA (Con siRNA) or EB1 siRNA 1 or 2 or EB3 siRNA 1 or 2 before seeding onto Matrigel in the Transwell insert. After 48 h, cells on the lower surface of the insert membrane were stained with cresyl violet and counted. EB3 knockdown has a greater effect on PC-3 invasion than EB1. Error bars are mean±s.e.m. from four independent experiments each of two replicates per condition. Significant differences: **P<0.01, ***P<0.001.