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. 2010 Jun 8;102(12):1724–1730. doi: 10.1038/sj.bjc.6605714

Figure 5.

Figure 5

The function of rebound pERK signalling in the escape from PLX4720 treatment. (A) U0126 blocks the rebound increase in pERK after PLX4720 treatment. Melanoma cells were either treated with vehicle (0), PLX4720 (3 μM) or PLX4720 + U0126 (both 3 μM) for 48 h, protein was then probed for expression of pERK and tERK. (B) Melanoma cells were treated with increasing concentrations of U0126 for 1 h before being probed for pERK and tERK expression. (C) Cells were treated with increasing concentrations of PLX4720 (30 nM–30 μM) for 24 h in the absence or presence of U0126 (3 μM), after which time, protein was extracted and resolved by western blotting and probed for either cleaved PARP (cl-PARP), phospho-ERK (pERK), cyclin D1 (Cyclin D1), p27 or cleaved caspase-3 (cl-casp-3). Blots were stripped once and probed for actin to show equal protein loading. (D) Combined BRAF and MEK inhibition leads to enhanced apoptosis. WM164 cells were treated with either vehicle, U0126 (3 μM, 3U0), PLX4720 (3PLX, 3 μM) or the two inhibitors in combination for 48 h. Levels of apoptosis were measured by annexin-V staining and flow cytometry. Data show the mean of three experiments. *P<0.05.