(A) Effect of vertical targeting of MAPK pathway on ERK activity as measured by phosphorylated ERK (pERK, Thr202/Tyr204)) and phosphorylated p90RSK (pRSK, Thr573). Apoptosis marker is also shown (cleaved PARP). Cells were treated with DMSO (control), 2 nM trametinib, 100 nM ERKi LY3214996, 100 nM pan-RAFi LY3009120 or combinations for 72 h and cell lysates analyzed by western blots. (B) Growth curves of RMS cells following treatment with DMSO, trametinib, ERKi LY3214996, pan-RAFi LY3009120 or combinations. Cells were plated in 96 well plates and next day treated as in A. Percent confluency was determined by imaging with Incucyte every 2 h for 168 h. (C) Crystal violet staining images of RAS-mutant RMS cell lines and one RAS wild-type (Rh18) upon treatment with trametinib, ERKi LY3214996, pan-RAFi LY3009120 or combinations. Cells were plated at low density in 12-well plates and next day treated with DMSO or increasing concentrations of the indicated drugs/combinations. After two weeks, cell growth was visualized with crystal violet staining.