Meki trametinib

SK‐MEL‐147 cells were lentivirally transduced with two GeCKO libraries (A and B) at a MOI of 0.3 aiming to ensure that most cells receive only one viral construct (Shalem et al., 2014). Briefly, 5 × 10⁶ cells were plated in 10 cm² dishes. 48 hr after infection, cells were selected with puromycin (1 µg/ml) for 14 days. Cells were split into two pools: One arm was subjected to 100 nM MEKi trametinib treatment (GSK1120210 Selleckchem), whereas the other arm was left untreated. Colonies formed in the drug‐treated arm were individually picked and expanded. For identification of sgRNAs in the individual clones, genomic DNA was isolated and sgRNAs were recovered by PCR amplification. Amplified DNA fragments were cloned into the TOPO TA‐cloning vector (450071, Invitrogen), followed by identification of the sgRNA by Sanger sequencing.

Chemicals used in this study (stocks resuspended in DMSO unless otherwise stated): BRAFi PLX4720 and PLX4032 (Selleck), BRAFi Dabrafenib (GSK2118436, ChemieTek), MEKi Trametinib (GSK1120212, Selleck), MEKi PD184352 (Selleck), MEKi AZD6244 (Selleck), ERKi SCH772984 (Selleck), ROCKi GSK269962A (Axon Medchem), ROCKi H1152 (resuspended in water; Calbiochem), AGC kinase inhibitor and ROCKi AT13148 (Selleck), myosin II inhibitor blebbistatin (in 95% DMSO; Calbiochem). Concentrations used unless otherwise stated in other STAR Methods sections: 5 μM ROCKi GSK269962A, 5 μM ROCKi H1152, 5 μM ROCKi AT13148, 25 μM myosin II inhibitor blebbistatin, 5 μM BRAFi PLX4720. “Analysis of cell morphology” section lists the inhibitors and concentrations used for those experiments.