EGFR-PI3K-AKT signaling cascade differentially effects loss of genetic NRAS in NRAS mutant melanoma cell lines. (A and B), Cell Titer MTS tetrazolium assay displaying mean cell viability of MELJUSO (A) and Hs944T (B) cell lines expressing GFP, AKT1, AKT2, PI3K, or EGFR with or without knockdown of NRAS (six replicates) after 96 h. (C and D), Representative images of crystal violet stained clonogenic growth assays with MELJUSO (C) and Hs944T (D) cell lines expressing GFP, AKT1, AKT2, PI3K, or EGFR with or without knockdown of NRAS (duplicate), each from three independent experiments. (E and F), Representative immunoblots displaying the effect of MELJUSO (E) and Hs944T (F) cell lines expressing GFP, AKT1, AKT2, PI3K, or EGFR with or without knockdown of NRAS, after 96 h on phosphorylated and/or total protein levels of NRAS, EGFR, PI3K, RB, AKT, ERK, and CCND1. β-actin immunoblotting was used to determine equivalent loading.