Figure 3. Nm23-H1 impairs Gelsolin- depolymerization activity.

A. A pyrene-actin polymerization assay was used to measure the actin-polymerization activity in protein lysates from 4T1 cells expressing either vector, Flag-tagged Nm23-H1 (Nm23-H1), GFP-tagged Gelsolin (Gelsolin) or both proteins (Nm23-H1/Gelsolin). Protein lysate was incubated for 2 hours in presence of pyrene-actin monomers and a polymerization buffer. The increase in fluorescence intensity at 590 nm was measured using VersaMax Microplate Fluorescence Reader and Softmax Pro 5.4 software to obtain the Vmax of each reaction. Data represent the ratio of Vmax of each sample versus Vmax of the vector transfectants. B. F-actin-depolymerization activity of the same samples was measured by incubating protein lysate in presence of pyrene-actin filaments for 2 hours. The decrease of fluorescence intensity at 590 nm was measured and Vmax ratio relative to the vector samples was graphed. C. Actin cytoskeleton structure in 4T1 cells was visualized by staining the cells with rhodamine-phalloidin (red) and DAPI (blue). 400X magnification images are shown. D. Actin-polymerization activity in protein lysates from MDA-MB-231T cells expressing either vector, Flag-tagged Nm23-H1 (Nm23-H1), GFP-tagged Gelsolin (Gelsolin) or both proteins (Nm23-H1/Gelsolin) was measured as previously described for Figure 3A. E. F-actin-depolymerization activity in protein lysates from MDA-MB-231T cells was determined as previously described for Figure 3B. F. Actin cytoskeleton structure in MDA-MB-231T cells was visualized by staining the cells with rhodamine-phalloidin (red) and DAPI (blue). 400X magnification images are shown. G. Western blot (left panel) and F-actin-depolymerization activity (right panel) of lysates from MCF7 expressing either non-target control shRNA (−) or shRNA specific to Nm23-H1 (+).