Figure 2. Activation of c-Abl and Arg promotes invasion, proliferation, and survival of melanoma cells.

(a) Serum-starved WM3248 cells, transfected with two independent cAbl or Arg siRNAs, were incubated in matrigel invasion chambers for 48h utilizing IGF-1 (10nM) as chemoattractant. The total number of invaded cells on the undersurface of the membranes were counted and expressed as a percentage of scrambled. Graphs are Mean ± SEM, n=3 independent experiments, performed in duplicate. **0.001≤p<0.01; ***p<0.001. Photographs are at 100X magnification. Knockdown of c-Abl and Arg was assessed by semi-quantitative RT-PCR (right). (b) Melanoma cells were serum-starved and treated with nilotinib (0.5μM) for 16–20h, and incubated in invasion assays as described in (a) with nilotinib in the top and bottom chambers. Graphs are Mean ± SEM, n=3, normalized to untreated. *p<0.05. (c,d) Tritiated thymidine incorporation was assessed in cells treated with STI571, nilotinib, or transfected with siRNAs. CPMs were normalized to vehicle-treated or scrambled-transfected cells. Mean ± SEM, n=3 independent experiments, performed in triplicate. Some error bars are too small to be visualized. (d, middle) The level of c-Abl/Arg activation and the number of nilotinib targets were added together to create a “Score” (Table 2, Fig. 1a, Fig. 2e), which was plotted against tritiated thymidine incorporation results. An inverse correlation between the “Score” (number of nilotinib targets) and the sensitivity to nilotinib was observed. R²=0.45, p=0.011. Lysates from untreated (e) or nilotinib-treated (24h) (d-bottom) cells in serum conditions were blotted with the indicated antibodies. (f) Lysates from detached and attached cells treated with vehicle or STI571 and deprived of serum for 72h (WM3248) or 96h (435s/M14) were probed with the indicated antibodies. Scr=scrambled.