(A) UM001, UM003 and UM004 cells were treated with 100 nM of trametinib (GSK1120212) for the indicated times. Cell lysates were probed with phospho ERK1/2, total ERK2 and actin antibodies. (B) UM001, UM003 and UM004 cells were treated with DMSO or trametinib for 3 days (UM001 and UM004 cells) or 5 days (UM003). Cells were then fixed, permeabilized and subjected to propidium iodide (PI) staining. Cell cycle analysis was performed with FlowJ software. *P<0.05, **P<0.01, ***P<0.001, based on two-tail Student's t-test assuming unequal variance. (C) UM001 cells were treated with vehicle control, 10 ng/ml of EGF, PDGF-B, HGF, NRG1 and IGF1 alone or together with 100 nM trametinib. After 72 hr, cells were subjected to crystal violet staining. Representative microscopic images of the cells at 200× magnification are shown. Scale bar is equal to 50 μm. (D) UM003 cells were treated as in C for a total of 5 days. Drugs and growth factors were replenished on day 3. Cells were stained with crystal violet.