Fig. 3.
Re-expression of ITF-2A in colon cancer cells attenuates colony formation. (A,B) DLD1/ITF-2B, DLD1/ITF-2A and HCT116/ITF-2A clones were plated in semi-solid agar medium and treated with doxycycline (Dox) for four weeks. Colonies were counted and results are expressed as means ± SD of an experiment performed in triplicates.
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Fig. 4.
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ITF-2A regulates CDKN1A transcription in a p53-independent manner
Previous experiments demonstrated that the Telaprevir inhibitor p21Cip1 is a crucial mediator of ITF-2B-induced cell cycle arrest [2]. To test whether ITF-2A is also capable of inducing CDKN1A transcription, DLD1 and HCT116 cells were transiently transfected with the CDKN1A reporter gene construct K/DA containing a CDKN1A promoter fragment with E boxes located in the first intron of the CDKN1A gene (Fig. 4B). In both cell lines, ITF-2A was capable of inducing this reporter gene construct (Fig. 4C and D). Mutation of two of these E boxes, namely E11 and E12, reduced the activity of the reporter gene constructs back to the level of the reference construct K, containing only a minimal CDKN1A promoter. These data clearly demonstrate that ITF-2A regulates CDKN1A transcription. Interestingly, ITF-2A is weaker transcriptional activator when compared to ITF-2B (compare Fig. 4C and E/D and F).
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