Research Paper Volume 7, Issue 11 pp 928—936

Figure 2. Molecular study of OTX2 responsive elements in the p63 gene. (A) The H1299 cell line has been transfected using OTX2 cDNA, and the expression of p63, p53 and p73 analyzed at 24 and 48 hours by RTqPRC analysis. The graphs show that OTX2 nicely induces the transcription of TAp63 isoform, with very low effects on p53 and none on TAp73, *t-test on DDCt values, coupled, two tails, *p = 0,0005, **p = 0,00073. (B) Induction control respect to DeltaN isoforms, the graph show that 48 hours and none of these isoform are transactivated. (C) ChIP analysis, showing the binding of OTX2 at the responsive element, located at the 5′ (upper panel), respect to that located within intron 5. In the first case the PCR product is detectable at cycle 31 whereas the other is only detectable later after 43 cycles. (D) qPCR analysis using DNA pulled down using OTX antibody. t-test on DDCt values, coupled, two tails, *p = 0,00057.( E) Western blot of protein extracted from cells after the transfection. A band corresponding to TAp63 molecular weight is visible. The control has been obtained using protein extracts from a SaOS TET-On cell line.