Figure 1. HGF promotes cell cycle progression of trametinib-treated UM cells.
(A) HGF promotes S-phase entry in trametinib-treated cells. 3.0 × 105 UM001 cells and UM004 cells were seeded in triplicates and treated with DMSO, 50 nM trametinib, 10 ng/ml HGF, and a combination of 50 nM trametinib and HGF, respectively, for a total of 32 hours. A final concentration of 10 µM EdU was allowed to incorporate for 16 hours before processing. S-phase entry is normalized to DMSO condition, and data points are indicative of 3 experimental repeats. *P<0.05, **P<0.01, ***P<0.001. (B) HGF inhibits trametinib-induced apoptosis in UM cells. UM001 and UM004 cells were treated with DMSO, 10 ng/ml HGF, 50 nM trametinib, a combination of HGF and trametinib, respectively, for 48 hours. Cells were then subjected to annexin V/PI staining. The percentage of annexin V-positive cells is graphed. *P<0.05, **P<0.01, ***P<0.001. (C) HGF modulates expression of cell cycle regulators in trametinib-treated cells. UM001 cells (left) and UM004 cells (right) were treated with 50 nM trametinib, 10 ng/ml HGF, 2.5 µM MK2206, or combinations as indicated for 48 hours. Cell lysates were probed for levels of cell cycle regulators cyclin A2, cyclin D1, phospho-RB, RB, cleaved PARP and cleaved caspase 3, respectively. Levels of pAKT and pERK were also determined by Western blotting.