FTI-277 does not affect the localization of PAKs and PhoPAKs in HeLa and A375MM cells. HeLa and A375 MM cells were treated for 48 h as indicated in Figure 2 and in Methods, and stained with relevant antibodies. Olympus ScanR analysis software was used to calculate the intensity of the fluorescent signal present in nuclei and in the whole cells. A. The graph represents the relative amount (%) of PAKs present in nucleus versus the amount present in the whole cell in the indicated treatment. B. The graph represents the relative amount (%) of PhoPAKs (respective panels) present in nucleus versus the amount present in the whole cell in the indicated treatment. C. The graph represents the relative amount of PAKs, PhoPAKs, and PhoPAK clusters in A375MM cells treated for 48 h as indicated in Figure 2 and in Methods. The mean intensity of the signal in vehicle samples was arbitrarily considered 100%. The amount of PhoPAK spots present in the nuclei of each sample was normalized against the total number of cells counted. All graphs (panel A, B, C) shows the mean ± SD of at least 2 independent experiments, each run in triplicate (three wells per condition). The statistical significance of the indicated treatments was calculated using t-test: ns = not significant, p-value >0.05; * significant, p-value < 0.05; **** = highest significance, p-value <0.0001.