Figure 2. Enhanced invadopodia formation in FAK-deficient MTLn3 cells requires MMP activity but is not sufficient for invasion. (A) MTLn3 cells transiently transfected with control siRNA (Ctlsi) or FAK siRNA (FAKsi) were cultured on fibronectin–Alexa Fluor 568 gelatin coverslips in the presence of DMSO vehicle control (−GM6001) and stained with anticortactin antibody (green). (B) MTLn3 cells transiently transfected with control siRNA or FAK siRNA were cultured on fibronectin–Alexa Fluor 568 gelatin-coated coverslips in the presence of 50 µM GM6001 (+GM6001) and stained with anticortactin antibody (green). Boxed regions in A and B depict regions of invadopodia shown magnified in insets. (C) Quantification of active invadopodia, defined as colocalizing areas of cortactin staining and matrix degradation, is expressed as the mean number of active invadopodia per cell. (D) MTLn3 cells transiently transfected with control siRNA or FAK siRNA were cultured in the presence of vehicle control (−GM6001) or 50 µM GM6001 (+GM6001) and were assayed for their ability to invade through Matrigel-coated membranes. Relative invasion was determined by normalizing the number of cells invaded to those transfected with control siRNA (−GM6001). Data shown are means ± SEM of three independent experiments. *, P < 0.05 by one-way ANOVA compared with control siRNA (−GM6001). Bars, 10 µm.
Figure 2.

Enhanced invadopodia formation in FAK-deficient MTLn3 cells requires MMP activity but is not sufficient for invasion. (A) MTLn3 cells transiently transfected with control siRNA (Ctlsi) or FAK siRNA (FAKsi) were cultured on fibronectin–Alexa Fluor 568 gelatin coverslips in the presence of DMSO vehicle control (−GM6001) and stained with anticortactin antibody (green). (B) MTLn3 cells transiently transfected with control siRNA or FAK siRNA were cultured on fibronectin–Alexa Fluor 568 gelatin-coated coverslips in the presence of 50 µM GM6001 (+GM6001) and stained with anticortactin antibody (green). Boxed regions in A and B depict regions of invadopodia shown magnified in insets. (C) Quantification of active invadopodia, defined as colocalizing areas of cortactin staining and matrix degradation, is expressed as the mean number of active invadopodia per cell. (D) MTLn3 cells transiently transfected with control siRNA or FAK siRNA were cultured in the presence of vehicle control (−GM6001) or 50 µM GM6001 (+GM6001) and were assayed for their ability to invade through Matrigel-coated membranes. Relative invasion was determined by normalizing the number of cells invaded to those transfected with control siRNA (−GM6001). Data shown are means ± SEM of three independent experiments. *, P < 0.05 by one-way ANOVA compared with control siRNA (−GM6001). Bars, 10 µm.

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