Figure 5.

Stx4 N-terminal peptide impairs invadopodium formation and gelatin degradation. A, invadopodium-based degradation of gelatin by cells transfected with GFP (Control), GFP-Stx4-FL, and GFP–Stx4–N-term. Cells were transfected for 20 h, seeded onto fluorescent gelatin for 4 h, and then fixed, permeabilized, stained for F-actin, and analyzed by confocal microscopy. Scale bars = 10 μm. B, quantification of invadopodium formation. Cells were transfected with the following: GFP alone, GFP-Stx4-FL, GFP–Stx4–N-term, GFP-Stx3 N-terminal peptide, GFP + control siRNA, GFP + Munc18c siRNA, GFP-Stx4-FL + siRNA Control, GFP-Stx4-FL + siRNA Munc18c, GFP–Stx4–N-term + siRNA control, and GFP–Stx4–N-term + Munc18c siRNA. Cells were transfected for 44 h and then seeded onto fluorescent gelatin and processed as in A. Cells with F-actin puncta overlying dark spots of gelatin degradation were counted as cells forming invadopodia. Percentages of cells forming invadopodia, normalized to GFP alone, were determined by counting 50 cells/sample. C, cells expressing either GFP, GFP-Stx4-FL, or GFP–Stx4–N-term were transfected for 24 h, seeded onto fluorescent gelatin for 24 h, and then fixed. Parental cells and cells expressing GFP were analyzed for dark areas of degradation and scored as described under “Experimental procedures.” All data are presented as percent of control ± S.D. Asterisks denote values significantly different from control (*, p < 0.05). All data represent three or more biological replicates with at least three technical replicates.