Fig 4.

Net1A is required for trailing edge retraction in MDA-MB-231 cells. (A) MDA-MB-231 cells were transfected with the siRNAs shown. Three days later, the monolayer was scratched and cells were allowed to migrate back into the cleared area. After 6 h, the cells were fixed and stained for DNA (blue) and F-actin (red). Arrows show the direction of migration. Shown are representative images from three independent experiments. Bar, 20 μm. (B) Representative Western blot of siRNA-transfected cells. (C) Quantification of elongation index in migrating MDA-MB-231 cells transfected with the siRNAs shown. The elongation index was measured as the distance from the furthest tip of the cell to the middle of the nucleus divided by the width of the cell at that portion of the nucleus. Shown are the combined results from three independent experiments. Bars denote median values. ***, P < 0.001. (D) Reexpression of Net1 proteins in control or Net1 dual isoform siRNA-transfected cells. Two days after siRNA transfection, cells were retransfected with expression vectors for Myc epitope-tagged, nucleus-localized β-galactosidase or the siRNA-resistant, HA-tagged Net1 isoforms shown. Net1A L/E, catalytically inactive Net1A L²⁶⁷E. Cells were allowed to migrate into a wounded area, fixed, and stained for the antigens shown. Arrows denote the direction of migration. Shown are representative images from three independent experiments. Bar, 20 μm. (E) Representative Western blot of siRNA-transfected cells. (F) Quantification of elongation indices from three independent rescue experiments. Bars denote median values. ***, P < 0.001. (G) Representative Western blot of transfected cells from elongation index rescue experiments.