Figure 2

FTI-277 treatment of HeLa cells up-regulates PAKs and phosphorylated PAKs. The statistical significance of the treatments was calculated using t-test: ns = not significant, p-value >0.05; * = significant, p-value <0.05; ** highly significant = p-value <0.01). n = n. of biological replicates. Each biological replicate consider the average mean intensity data obtained from analysis of three wells per condition. A. Fluorescently stained HeLa cells treated for 48 h with the indicated concentration of FTI-277 or DMSO (Vehicle). Panels: PAK = anti-PAK (C19) antibody; Hoechst = nuclear staining; Merge = Hoechst + PAK; Mask: software mask that identifies nuclear PAKs (green), and PAK clusters (spots) within nuclei (red). scale bar =10 μm. B. The graph shows the relative amount (%) of PAKs in the nuclei in 4 h FTI277-treated cells relative to vehicle, arbitrarily set as 100%. C. The graph shows the relative amount (%) of total PAKs in 48 h FTI-277-treated cells compared to vehicle-treated cells; D. Immunoblot analysis of total lysates from HeLa cells treated as indicated in C. E. Fluorescently stained HeLa cells treated for 48 h with the indicated concentration of FTI-277 or DMSO (vehicle). Panels: PhoPak = α-Phospho-PAK1/2/3[pThr423] antibody; Hoechst = nuclear staining; Merge = Hoechst + PhoPak; Mask: software mask that identifies nuclear PhoPaks (green), and PhoPak clusters (spots) within nuclei (red). F. The graph shows the relative amount (%) of total PhoPaks in 4 h FTI277-treated cells relative to vehicle-treated cells. G. The graph shows the relative amount of total PhoPaks in 48 h FTI277-treated cells compared to vehicle-treated cells; H: immunoblot analysis of total lysates form HeLa cells treated as in G. I. The graph shows the relative number (%) of PhoPaks spots within the nuclei in 48 h FTI277-treated cells relative to vehicle-treated cells, which were arbitrarily considered 100%.