Fig. 6

GroPIns facilitates Shp1 localization and association with cortactin at invadopodia. A Representative images of PLA assay carried out on A375MM cells cultured on fluorescent gelatin-coated coverslips and then treated with 50 μM GroPIns for 1 h (see “Methods” section). Cells were fixed and stained with antibodies against endogenous Shp1 (rabbit polyclonal antibody) and endogenous cortactin (monoclonal murine antibody), followed by incubation with PLA probes minus and plus, ligation and amplification. PLA signal (red spots) indicates Shp1-Cortactin complexes. B Quantification of PLA signal/Degradation area colocalization (expressed as percentages of control) of cells treated as in A. Data are expressed as the means (± SE) of two independent experiments performed in duplicate. C Representative confocal images of A375MM cells grown on fluorescent gelatin-coated coverslips for 16 h and then untreated (Ctr) or treated with 50 µM GroPIns for 1 h. The cells were fixed and stained for shp1 and cortactin (following cytosol extraction as described in “Methods” section). Shp1, cortactin and gelatin are shown in green, magenta and gray, respectively. The degraded ECM is shown as dark areas and refers to the degradative activity resulting from ~ 17 h of incubation of cells on the gelatin. Invadopodia present following 1 h of GroPIns treatment are shown as actin-positive dots corresponding to the dark areas. Arrowheads indicate invadopodia colocalizing with Shp1. D Quantification of invadopodia colocalizing with Shp1 (expressed as percentage of total cells) of cells treated with 50 µM GroPIns for the indicated times. Data are expressed as the means (± SE) of three independent experiments performed in duplicate. ***P < 0.001; *P < 0.05 (Student’s t-test) calculated for each treatment versus untreated samples. Scale bars, 10 μm