Fig. 3.

Activation of NGF-TrkA signaling induces cell cycle arrest of MM cells. a and b, representative images showing the morphology of MM cells upon TrkA ectopic expression and following activation by NGF. Stably transduced cells with either doxycycline-inducible vector (SK-MEL-28-TrkA and G-361-TrkA) or empty vector (SK-MEL-28-E and G-361-E) were incubated for 48 h with or without doxycycline (500 ng/ml) and next treated with NGF (100 ng/ml) or vehicle for 24 h in 2 % FBS medium. Images were obtained using phase-contrast microscopy from four independent experiments. Arrowheads indicate cells displaying distinctive morphological changes of oncogene-induced type growth arrest of intracellular vacuolization and cell shrinkage. c and d, cell proliferation of SK-MEL-28-E/TrkA and G-361-E/TrkA was tested by cell-cycle assay performed using DNA staining with propidium iodide and FACS analysis. Cells were incubated with or without doxycycline (500 ng/ml) for 48 h and then stimulated with NGF (100 ng/ml) for 24 h or vehicle treated. Bar graphs represent frequency of cell-cycle phases as the mean ± SD of n = 3 independent replicates (Student’s t test: *, p < 0.05 compared to vehicle control). Dox, doxycycline