Figure 2

Drebrin and pS142-drebrin are upregulated in human prostate cancer cell lines. (a) Immunoblots of human prostate epithelial cell lines probed with antibodies against drebrin, pS142-drebrin, Cdk5, p35 and, as a loading control, GAPDH. Drebrin is expressed at moderate levels in the normal human prostate epithelial cell line PNT2-C2 and the human prostate cancer cell line DU-145, and at higher levels in the human prostate cancer cell lines LNCaP and PC-3. There is a tight correlation between the levels of drebrin and pS142-drebrin. p35 is highly upregulated in prostate cancer cell lines compared to PNT2-C2 cells. (b) Quantification of relative protein levels from immunoblots of PNT2-C2, LNCaP, DU-145 and PC-3 cell lysates probed with antibodies against drebrin, pS142-drebrin, Cdk5 and p35. Error bars are mean±s.e.m. from at least three independent experiments. Significant differences: *P<0.05; **P<0.01. (c–e) Immunofluorescence images of PC-3 cells in 3D Matrigel in a CXCL12 gradient in chemotaxis chambers. The source of CXCL12 is to the left. Cells were labelled with antibodies against drebrin and tyrosinated α-tubulin (tyr-tubulin), to identify dynamic microtubules, and with phalloidin, to label F-actin. (c) An unpolarized, rounded cell with F-actin containing filopodia extended in all directions (arrowheads). Drebrin is distributed in the cell cortex co-localizing with F-actin. (d) A polarized cell before pseudopod production. The longest filopodia are extended on the side of the cell facing the CXCL12 gradient (arrowheads). Dynamic microtubules (tyr-tubulin) have entered the filopodia facing the gradient and drebrin has re-located to their base. (e) A polarized cell with several pseudopods (asterisks) covered in filopodia (arrowheads). Drebrin strongly co-localizes with F-actin, particularly at the base of filopodia (arrowheads). Large bundles of dynamic microtubules (tyr-tubulin) enter pseudopods and single or small bundles of microtubules enter some filopodia (curved arrow).