Fig. 2

Shp1 regulates invadopodia dynamics in A375MM cells. A Representative confocal images of non-targeting siRNAs and Shp1 siRNAs-treated A375MM cells plated on fluorescent gelatin-coated coverslips. Cells were then fixed and stained with phalloidin (magenta). B Western blot analysis of Shp1 protein in A375MM cells upon Shp1 knock-down. Actin was used as a loading control. Molecular weight standards (kDa) are indicated on the left the panel. C, D Quantification of cells forming invadopodia (expressed as percentage of total cells; at least 80 cells were analyzed for each condition) (C) and of the total area of degradation (expressed as percentages of control; at least 30 cells were analyzed for each condition) (D) of cells treated as in A. (E) Representative confocal images of A375MM cells transfected with Shp1 WT or the Shp1 C455S mutant and plated on fluorescent gelatin-coated coverslips (gray). Cells were fixed and stained with an anti-Shp1 antibody (green) and phalloidin (magenta). F, G Quantification of cells forming invadopodia (expressed as percentage of total cells; at least 80 cells were analyzed for each condition) (F) and of the total area of ECM degradation (expressed as percentages of control; at least 30 cells were analyzed for each condition) (G) of cells treated as in E. Degraded ECM is shown as dark areas on gray gelatin. The invadopodia are shown as actin-positive dots corresponding to the dark areas. Arrowheads indicate active invadopodia. Data are expressed as the means (± SE) of at least three independent experiments performed in duplicate. ***P < 0.001; **P < 0.02; ns P > 0.05 (Student’s t-test) calculated for each treatment versus untreated samples. Scale bars, 10 μm