Depletion of NIR may activate p53 by de-repressing p53 acetylation.

<p>A. U2OS cells were transfected with NIR-specific siRNA or control siRNA respectively. At 72 hrs post transfection, cell lysates were prepared. Proteins from the lysates were separated on SDS-PAGE, transferred onto PVDF membrane. Blot was probed for detection of NIR, p53 and p21. Topoisomerase I (Topo I) was used as a loading control. RT-PCR was performed with RNA extracted from the above siRNA transfected cells for evaluation of p21 mRNA. Amplification of GAPDH was used as a loading control. B. U2OS cells were transfected with NIR-specific siRNA or control siRNA respectively. At 72 hrs post transfection, cell lysates were prepared. Immunoprecipitation was performed with anti-acetyl-lysine antibody on the cell lysates. Proteins from the immunoprecipitates were separated on SDS-PAGE and transferred onto a PVDF membrane. Blot was probed with anti-p53 or anti-UBF antibody. C. U2OS cells were transfected with NIR-specific siRNA or control siRNA respectively. At 72 hrs post transfection, cell lysates were prepared. Proteins from the lysates were separated on SDS-PAGE, transferred onto PVDF membrane. Blot was probed for detection of NIR, PUMA and BAX. Beta-actin was used as a loading control. D. U2OS cells were transfected with NIR siRNA or control siRNA respectively. Two thousand cells were seeded in 6 mm plates 72 hrs post-transfection. Cells were grown for 10 days and cell colonies were stained with Coomassie Bright Blue after fixation with ethanol/acetic acid. E. U2OS cells were transfected with NIR siRNA or control siRNA respectively. Cells were seeded in 96-well plates 72 hrs post transfection. Ten microliters of CCK-8 was added to the cells and absorbance at 450 nm was measured at different time periods. The experiment was repeated three times in duplicate and growth curves were plotted using absorbance at 450 nm vs. different time points.