Fig. 4: PANDAR disturbed the binding of p53 protein with CDKN1A promoter.

a Schematic diagram showing the experimental design of purified PANDAR competitively binding with purified p53 protein at the CDKN1A promoter. We used 0.4 μg purified p53 protein and incubated it with 1 μg biotinylated CDKN1A promoter. After incubation, purified PANDAR or control probe U6 was added to the reaction mixture to compete with the p53:CDKN1A complex. Immunoblotting analysis was used to detect the residual amount of p53 protein after biotin precipitation. b Amount of purified p53 protein, purified PANDAR and control probe U6. M, maker. c Different amount of purified PANDAR and control probe U6 competing with the p53:CDKN1A complex. d The imageJ software was used to quantitate the residual amount of p53 protein after biotin precipitation. e Schematic diagram showing the modified experimental design of total RNA containing PANDAR competitively binding with purified p53 protein at the CDKN1A promoter. After incubation, different amounts of total RNA extracted from cancer cells were added to the reaction mixture to compete with the p53:CDKN1A complex. Immunoblotting analysis was used to detect the amount of p53 protein after biotin precipitation. f Amount of PANDAR in total RNA was determined by qRT-PCR. g Different amounts of total RNA containing PANDAR competed with the p53:CDKN1A complex. h Amount of PANDAR was determined by qRT-PCR in cancer cells treated with the CRISPR/Cas9 system for PANDAR. i Depletion of PANDAR in total RNA failed to compete with the p53:CDKN1A complex. j ChIP assays with an anti-p53 or negative control (anti-IgG) antibodies showed that the binding of p53 protein with the CDKN1A promoter was regulated by PANDAR in human GC cells. The y-axis represents the % input of the promoter fragments captured by the two different antibodies