Fig 1: HME1-shp53 cells can be transformed by all four RAS-G12V mutant isoforms, but not by their wild-type counterparts. (a) Illustration of the scheme for creation of the HME1-shp53 cell line by sequential addition of SV40 small T antigen, shRNA against p53, and ER-tagged wild-type or G12V-mutant RAS isoforms into HME1-hTERT cells.26, 27 The parental HME1-hTERT (ATCC CRL-4010™) cells were obtained from American Type Cell Culture (ATCC) and mycoplasma-free. Stable expression of genes was achieved by transduction with retroviral expression vectors pBABE-hygro (for SV40 Small T Antigen), pRetroSuper (for shRNA against p53) or pBABE-puro (for all forms of RAS)28, 29 with viruses generated via calcium phosphate transfection,30 followed by stable selection using 20 μg/ml hygromycin for pBABE-hygro (Invitrogen, Carlsbad, CA, USA), 5 μg/ml blasticidin for pRetroSuper (Sigma Aldrich, St Louis, MO, USA), and 5 μg/ml puromycin (Sigma Aldrich) for pBABE-puro. Ras isoforms were cloned using primers listed in Supplementary Table S1. G12V mutations were introduced using QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies) and the targeting oligonucleotides are listed in Supplementary Table S1. (b) HME1-shp53 cells expressing wild-type or ER-tagged G12V mutant of KRAS4A, KRAS4B, HRAS and NRAS as shown in (a) were cultured in soft agar in the presence or absence of 500 nM tamoxifen (Sigma Aldrich) for 14 days, and subsequently stained using methylthiazolyldiphenyl-tetrazolium bromide (MTT; Sigma Aldrich). This three-dimensional anchorage-independent soft agar culture was performed according to a previously published protocol25 supplemented with 0.5 μg/ml hydrocortisone (Sigma Aldrich), and 5 μg/ml insulin (I-DNA Biotech, Singapore). Using an Olympus SZX16 stereo microscope with 3.2 × zoom and 0.5 × objective, pictures of 4–5 random microscopy fields were taken per well after MTT staining; one representative field for each condition from one representative experiment out of three performed is presented. (c) Immunoblot study of cell lysates from HME1-shp53 control cells and those expressing RAS proteins. The cell lysates were prepared from the same cells used for the soft agar study in (b) and HME1-shp53 was used as the negative control. Immunoblots were done on cell lysates per standard protocol established in the laboratory.12 The antibodies for pan-RAS (Ras10, MA1-012) and GAPDH (14C10, #2118) were from Pierce Scientific (Waltham, MA, USA) and Cell Signaling Technology (Danver, MA, USA), respectively. Results presented in (b) and (c) are from a single experiment that is representative of three similarly performed studies.