Fig. 5

Proliferation arrest promoted by NGF-TrkA signaling in MM cells is enhanced after AKT pathway inhibition and dampened by the inhibition of MAPK pathway. a and b, stably transduced MM cells with doxycycline-inducible TrkA-vector (SK-MEL-28-TrkA and G-361-TrkA) were incubated for 48 h in 2 % FBS medium with doxycycline (500 ng/ml) and next treated with vehicle (DMSO), MAPK pathway inhibitor U0126 (5 μM), AKT pathway inhibitor LY294002 (25 μM), or the broad range receptor kinase inhibitor CEP-701 (10 μM), in the presence or absence of NGF (100 ng/ml) for 24 h in 2 % FBS medium. Images of morphological changes induced by treatment are representative of three independent experiments and were obtained using phase-contrast microscopy from cells growth in 96-well plate. c and d, cell proliferation or apoptosis of 48 h doxycycline-induced (500 ng/ml) SK-MEL-28-TrkA and G-361-TrkA cells, was measured 24 h post-treatment with NGF or indicated kinase inhibitors (as above), by using Click-iT EdU cell-proliferation assay or caspase-3 immunostaining, respectively. Images were acquired and quantified by the Operetta High Content Imaging System and cell count reported as fraction over total cell number (mean ± SD of n = 3 independent replicates; Student’s t test: **, P < 0.01; ***, P < 0.001). e and f, proliferation of SK-MEL-28-TrkA and G-361-TrkA cells, pre-induced with doxycycline (500 ng/ml) for 48 h in 2 % FBS medium, was monitored real-time by using the xCELLigence system. Cells were then maintained for 48 h in 2 % FBS medium with NGF (100 ng/ml) in the presence or absence of kinase inhibitors U0126 (5 μM), LY294002 (25 μM), CEP-701 (10 μM) and compared to vehicle control. Relative cell proliferation was measured by cell index (CI) and normalized at the beginning of treatment. Error bars are showed as SD below the trend-line of the mean from three independent biological replicates, each consisting of two internal replicates. One-way ANOVA was performed followed by Tukey’s post-test (***, P < 0.001). Dox, doxycycline