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Fig. 1 | Genome Medicine

Fig. 1

From: Linking FOXO3, NCOA3, and TCF7L2 to Ras pathway phenotypes through a genome-wide forward genetic screen in human colorectal cancer cells

Fig. 1

A piggyBac transposon screen in human CRC cells identifies putative Ras pathway genes. a, b Gene trap as well as promoter-containing piggyBac transposons can revert human CRC cells deprived of their oncogenic Ras pathway allele to survive growth in low glucose. Co-transfection of DLD-1 KRASwt/- and RKO BRAFwt/-/- cells with transposase (HyPBase) and gene trapping (PB-GT) or promoter-containing (PB-CAG-SD) transposon constructs was followed by selection in DMEM with 0.4 mM L-glucose for three weeks. Clones emerging after glucose deprivation were selected in hygromycin for 12 days to enrich clones with productive transposon integrations (in-frame gene trap or promoter insertion) and the surviving clones were stained and counted. Mean and standard deviation (SD) from three independent experiments. c, d Transposon mutagenesis and selection in low glucose results in upregulation of GLUT1, a phenotype of Ras pathway activation in human CRC cells. RT-PCR of GLUT1 in parental DLD-1 and RKO cells, their isogenic derivatives DLD-1 KRASwt/- and RKO BRAFwt/-/- with single clones and clone pools derived from mutagenesis with gene trap (PB-GT) or promoter-containing (PB-CAG-SD) transposons. Mean RQs and ΔCt SEs as fold of DLD-1 KRASwt/- and RKO BRAFwt/-/- from three replicates. The P < 0.05 values were calculated using Student’s t-test where ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05

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