Ulation and Cell MigrationFigure 5. Regulation of tumor cell invasion by p114RhoGEF. (A,B) MDA-MB-231 cells were transfected as indicated and invasion across Matrigel covered filter inserts was then analyzed. Panel A shows images of the matrix after crystal violet staining. Panel B shows quantifications of matrix associated cells (shown are means 6 1SD, n = 7). (C-E) Cells transfected as indicated were plated into 3D matrices and migration as analyzed by time-lapse microscopy for 5 hours. Panel C shows still images and panel D quantification of migration distances of cells plated in collagen or Matrigel, and panel E migration in the presence of the metalloproteinase inhibitor GM6001. Quantifications show means 23727046 6 1SD of four different fields (12 cells were analyzed for each field). doi:10.1371/journal.pone.0050188.gdid not have clear effects on double MLC phosphorylation. Quantification of such images confirmed that p114RhoGEF depletion led to a strong reduction of cells with cortical double phosphorylated MLC in cells on all matrices except fibronectin, on which the effect was only small and migration was not p114RhoGEF dependent (Fig. 4C and 6D). p114RhoGEF thusstimulates cortical myosin activation in single cells in a matrixdependent manner. The f-actin staining further confirmed the morphological change of the cells upon p114RhoGEF depletion and the cells started to form the typical leading edge structures of cells during mesenchymal-like movement on 2D matrices (Fig. 6C). This wasCortical Myosin Regulation and Cell MigrationFigure 6. Regulation of cortical myosin Enasidenib chemical information phosphorylation by p114RhoGEF. (A) MDA-MB-231 cells were extracted and p114RhoGEF was immunoprecipated. The precipitates were then analyzed by immunoblotting for the GEF, myosin IIA, and ROCKII. (B) MDA-MB-231 cells that had transfected with the siRNAs indicated were lyzed and subjected to immunoblotting using 1531364 the indicated antibodies. (C-G). Cells, transfected and plated as indicated, were fixed and then processed for immunofluorescence microscopy using antibodies against the proteins indicated. Panel C2 shows a larger magnification of the control and p114RhoGEF siRNA transfected cells plated on Matrigel shown in panel C1. Panel D shows a quantification of the percentage of cells with cortical staining for double phosphorylated myosin (shown are means 6 1SD). (C1, G) Bars, 10 mm; (C2, E, F) Bars, 5 mm. doi:10.1371/journal.pone.0050188.gfurther confirmed by staining for atypical Protein Kinase C and myosin IIA, which accumulated at leading edges in a more polarized manner in p114RhoGEF depleted cells (Fig. 6E). p114RhoGEF itself localized to the cell cortex in a patchy manner, reminiscent of the double phospho-MLC staining. Similar to polarized epithelial cells, a considerable fraction of p114RhoGEF was cytosolic, and both cortical and cytosolic staining disappeared when p114RhoGEF was depleted (Fig. 6F). The strong reduction in cortical double phosphorylation of MLC was surprising as p114RhoGEF depleted cells were still ableto move, albeit more slowly. As the single phosphorylated form was not affected by immunoblotting (Fig. 6B), we also stained the cells for phospho-MLC (S19), which is less active at suboptimal actin concentrations in vitro [14]. Strikingly, depletion of p114RhoGEF did not Pinometostat site detectably affect the staining, indicating that p114RhoGEF preferentially stimulates double phosphorylation of MLC (Fig. 6G). Given the strong effect on double phosphorylation of MLC o.Ulation and Cell MigrationFigure 5. Regulation of tumor cell invasion by p114RhoGEF. (A,B) MDA-MB-231 cells were transfected as indicated and invasion across Matrigel covered filter inserts was then analyzed. Panel A shows images of the matrix after crystal violet staining. Panel B shows quantifications of matrix associated cells (shown are means 6 1SD, n = 7). (C-E) Cells transfected as indicated were plated into 3D matrices and migration as analyzed by time-lapse microscopy for 5 hours. Panel C shows still images and panel D quantification of migration distances of cells plated in collagen or Matrigel, and panel E migration in the presence of the metalloproteinase inhibitor GM6001. Quantifications show means 23727046 6 1SD of four different fields (12 cells were analyzed for each field). doi:10.1371/journal.pone.0050188.gdid not have clear effects on double MLC phosphorylation. Quantification of such images confirmed that p114RhoGEF depletion led to a strong reduction of cells with cortical double phosphorylated MLC in cells on all matrices except fibronectin, on which the effect was only small and migration was not p114RhoGEF dependent (Fig. 4C and 6D). p114RhoGEF thusstimulates cortical myosin activation in single cells in a matrixdependent manner. The f-actin staining further confirmed the morphological change of the cells upon p114RhoGEF depletion and the cells started to form the typical leading edge structures of cells during mesenchymal-like movement on 2D matrices (Fig. 6C). This wasCortical Myosin Regulation and Cell MigrationFigure 6. Regulation of cortical myosin phosphorylation by p114RhoGEF. (A) MDA-MB-231 cells were extracted and p114RhoGEF was immunoprecipated. The precipitates were then analyzed by immunoblotting for the GEF, myosin IIA, and ROCKII. (B) MDA-MB-231 cells that had transfected with the siRNAs indicated were lyzed and subjected to immunoblotting using 1531364 the indicated antibodies. (C-G). Cells, transfected and plated as indicated, were fixed and then processed for immunofluorescence microscopy using antibodies against the proteins indicated. Panel C2 shows a larger magnification of the control and p114RhoGEF siRNA transfected cells plated on Matrigel shown in panel C1. Panel D shows a quantification of the percentage of cells with cortical staining for double phosphorylated myosin (shown are means 6 1SD). (C1, G) Bars, 10 mm; (C2, E, F) Bars, 5 mm. doi:10.1371/journal.pone.0050188.gfurther confirmed by staining for atypical Protein Kinase C and myosin IIA, which accumulated at leading edges in a more polarized manner in p114RhoGEF depleted cells (Fig. 6E). p114RhoGEF itself localized to the cell cortex in a patchy manner, reminiscent of the double phospho-MLC staining. Similar to polarized epithelial cells, a considerable fraction of p114RhoGEF was cytosolic, and both cortical and cytosolic staining disappeared when p114RhoGEF was depleted (Fig. 6F). The strong reduction in cortical double phosphorylation of MLC was surprising as p114RhoGEF depleted cells were still ableto move, albeit more slowly. As the single phosphorylated form was not affected by immunoblotting (Fig. 6B), we also stained the cells for phospho-MLC (S19), which is less active at suboptimal actin concentrations in vitro [14]. Strikingly, depletion of p114RhoGEF did not detectably affect the staining, indicating that p114RhoGEF preferentially stimulates double phosphorylation of MLC (Fig. 6G). Given the strong effect on double phosphorylation of MLC o.
