Figure 2. NOS3 is direct transcriptional target of p53.

1. A putative p53 binding site was identified in the NOS3 promoter using Genomatix, MatInspector module. NOS3-p53RE lies between −363 and −341 bp on the 602-bp NOS3 promoter.
2. pNOS3p-luc1 (NOS3 602-bp promoter luciferase construct) was transfected in L6 cells and the effect of p53 on luciferase activity was measured. Resveratrol was used to activate p53 protein (Western blot). p53 gene-activation induces a 14-fold increase in pNOS3p-luc1 luciferase activity (red bar). p53 gene-silencing using p53 siRNA reverses this effect. Data represent mean ± SD of six independent measurements. *p = 2.0E−12. The results show that p53 transcriptionally regulates NOS3 promoter.
3. pNOS3p-luc2, the NOS3-p53RE (−363 to −341) cloned in luciferase vector was transfected in L6 cells in presence of resveratrol to activate p53 protein. Results show that p53 induces a 16-fold increase in the NOS3-p53RE luciferase activity (red bar). p53 gene-silencing and p53 transcriptional inhibition using p53 siRNA and pifithrin-α abolishes the increase in luciferase activity. Data represent mean ± SD of six independent measurements. *p = 8.0E−12. The results show that p53 regulates NOS3 promoter via NOS3-p53RE. The mmpNOS3p-luc2 construct (mmp-mutant minimal promoter) with mutated sequence of NOS3-p53RE was transfected in resveratrol-treated L6 cells. No increase in the luciferase activity is observed, showing the specificity of NOS3-p53RE.
4. Interaction of p53 with NOS3-p53RE is confirmed using EMSA, results show presence of shift and super-shift, p215'RE is used as a positive control.
5. Chromatin immunoprecipitation (ChIP) was conducted in resveratrol-treated L6 cells to confirm in vivo binding of p53 to NOS3-p53RE. p53 shows an increase in NOS3-p53RE binding with increasing dose of resveratrol. Scrambled primers for PCR were used as negative control.