(a) NMac1 increases the NDPK activity of recombinant human Nm23-H1 in a concentration-dependent manner. NDPK activity was measured by the amount of ATP produced from 5 ng of Nm23-H1 with 5 μM ADP & UTP as substrates in 1 min with the indicated concentrations of NMac1 or 1% DMSO as vehicle. All experiments were performed in triplicate, and the data are expressed as the means ± S.D. (b) NMac1 reduces membrane ruffles. (Left) Localization of F-actin was analyzed by confocal microscopy. MDA-MB-231 cells treated with 25 µM NMac1 (or 0.05% DMSO as the vehicle) for 16 h were stained with rhodamine phalloidin. (Right) The number of cell ruffles was quantified by determining the ruffling index. (c) NMac1 reduces Rac1 activation. An active Rac1 pull-down assay was conducted with MDA-MB-231 cells at the indicated concentrations for 16 h. A representative experiment of three independent experiments is shown. (d) MDA-MB-231-Luc-D3H2LN cells were orthotopically injected into NOD/SCID mice, and the mice were treated with vehicle or NMac1 (N = 7 for the Veh group, N = 6 for the NMac1 group). (Left) Bioluminescence images showing metastatic tumor cells. (Right) Quantification of the bioluminescence images 3 weeks after NMac1 treatment. The data are presented as the mean total photon flux per second ± S.D.