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Figure 1

From: Transcriptional activity mediated by β-CATENIN and TCF/LEF family members is completely dispensable for survival and propagation of multiple human colorectal cancer cell lines

Figure 1

HT29 and HCT116 CRC cells survive and can be propagated in the complete absence of TCF/LEF expression. (a) Schematic representation of the TCF7 and TCF7L1 genes and their protein products. Exons are depicted as numbered boxes. Exons chosen for CRISPR/Cas9-induced deletion are shown in red. The locations of sgRNA target sites are indicated by red arrowheads. The position of start codons (AUG) and functionally important domains of the TCF7 and TCF7L1 proteins are marked and connected to the exon(s) by which they are encoded. HMG-box DNA-binding domain, NLS nuclear localization sequence, Grg Groucho-related gene, TLE Transducin-like enhancer of split, CtBP Carboxy-terminal binding protein, BD binding domain. (b,c) Western blot analyses to detect TCF/LEF expression in whole cell lysates from HT29 (b) and HCT116 cells (c) with the genotypes indicated. WT wild type, SKO single knock-out, biallelic inactivation of TCF7L2, DKO double knock-out, HT29 cells with biallelic inactivation of TCF7 and TCF7L2, TKO triple knock-out, HCT116 cells with biallelic inactivation of TCF7, TCF7L1, and TCF7L2. LoVo cells were included as positive control for TCF/LEF detection. α-TUBULIN (αTUB) was used to demonstrate equal loading. Molecular weights are given in kDa. Representative results from one of three independent biological replicates are shown. Full size/uncropped versions of the Western blot images are shown in Supplementary Fig. S7. (d,e) The bar plots summarize and compare RNA expression levels of TCF/LEF family members determined by qRT-PCR in HT29 (d) and HCT116 cells (e) with the genotypes indicated. LoVo cells were included as positive control. TCF/LEF transcript levels were normalized to those of GAPDH, and are displayed as relative expression (rel. expr.). Each dot represents an individual measurement. Error bars indicate SD (n = 3). Statistical significance of gene expression differences was not analyzed for this series of experiments due to different group sizes of WT, SKO, DKO and TKO cell clones (see “Methods”).

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