Fig 1: Eliminating ICMT function abolishes the transforming ability of G12V-mutant RAS. (a) Schematic illustration showing the creation of HME1-shp53-ICMT-wild type, ICMT-null 1cr and ICMT-null 4cr cells. Wild-type ICMT expression was eliminated from HME1-shp53 by creating frameshift mutations in the genomic Icmt loci using the CRISPR-Cas9 method via expression vector pSpCas9 (BB)-2A-GFP (Addgene #48138).31 Control empty vector pSpCas9 (BB)-2A-GFP (EV), as well as the same vector carrying guide RNA sequences (termed 1cr and 4cr, Supplementary Table S2) were introduced into cells by electroporation using Gene Pulser Xcell™ (Bio-Rad, Hercules, CA, USA). Single cells expressing GFP were individually sorted using BD FACSAria™ III (BD Biosciences, Franklin Lakes, NJ, USA) into 96-well plates for clonal expansion and subsequently screened for genomic frameshift mutations by fluorescent PCR-capillary gel electrophoresis using a protocol by Ramlee et al.32 Standard PCR and Sanger sequencing were performed to identify the presence of frameshift mutations in exon 1. (b) Schematic illustration of the predicted polypeptide translated from each allele of HME-1 shp53 cells expressing wild-type ICMT,21 as well as of truncated polypeptides translated from mutant alleles in individual HME-1 shp53 ICMT-null clones. Green, cytoplasmic motifs; pink, endoplasmic reticulum luminal motifs; yellow, transmembrane domains; white, frameshifted nonsense polypeptide sequences resulting from CRISPR/Cas9 action; asterisk (*), end of polypeptide chain. The sequences of the mutated alleles are shown in Supplementary Table S2. (c) Immunoblot study of lysates of HME1-shp53-ICMT wild type, ICMT-null 1cr, and ICMT-null 4cr cells expressing ER-tagged G12V-mutant KRAS4A, KRAS4B, HRAS or NRAS. These cells were cultured in the presence or absence of tamoxifen for 48 h, then harvested for lysate preparation for immunoblot analysis. Lysates of cancer cell lines (MiaPaCa-2, MDA-MB-231, NCI-H1299 and T24; all obtained from ATCC and mycoplasma-free) were also blotted. Beta-actin antibodies (AM1829B) were from Abgent (San Diego, CA, USA). (d) ICMT-wild-type or ICMT-null HME1-shp53 cells expressing ER-tagged mutant RAS isoforms or the pBABE-puro empty vector control were grown in soft agar in the presence or absence of tamoxifen for 14 days, and subsequently stained using MTT. The image for each condition is a representative microscopic field of three fields taken from each experiment. Results presented in (c) and (d) are from a single experiment that is representative of three similarly performed studies. For (c) and (d), the tamoxifen concentrations used were 500 nM (empty vector control, KRAS4A and KRAS4B), 100 nM (NRAS) and 25 nM (HRAS).