Skip to main content
. 2014 Jan 10;42(6):3565–3579. doi: 10.1093/nar/gkt1371

Figure 7.

Figure 7.

NIR can cooperate with MDM2 to inhibit p53. (A) NIR supports the inhibition of p53-mediated reporter gene transactivation by MDM2 in H1299 cells. Cultures were transfected with reporter plasmid PG13-luc (0.3 µg) containing 13 copies of a p53 response element in front of a luciferase gene. Cotransfection with p53 plasmid (5 ng) resulted in robust luciferase activity (25-fold activation). MDM2 plasmid and Flag-NIR plasmid were included in the transfections at 75 and 50 ng, respectively. T bars denote standard deviations derived from three experiments; P-values were calculated with student’s t-test. (B) NIR supports the inhibition of p53-mediated reporter gene transactivation by MDM2 in U2OS cells. Cultures were transfected and analysed like the H1299 cultures in (A). Since U2OS cells express wild-type p53, transfection with reporter-vector-only sufficed to produce strong luciferase activity, which was arbitrarily set as 1. T bars denote standard deviations derived from three experiments; P-values were calculated with student’s t-test. (C) Same experiment as in (B), with additional p53 plasmid (5 ng) to increase luciferase activity. MDM2 and Flag-NIR plasmids were used as before. T bars denote standard deviations derived from three experiments; P-values were calculated with student’s t-test. (D) Knockdown of endogenous NIR and MDM2 cooperate to stimulate transcription of the p53-responsive p21 gene. HCT116 cells were transfected with control siRNA (C), MDM2 siRNA (M), NIR siRNA (N) or M+N combined. After 48 h, total RNA was prepared and subjected to RT-qPCR for the quantitation of NIR (upper diagram), MDM2 (central diagram) and p21 transcripts. The lower diagram (grey bars) shows the p21 transcript levels in response to NIR and MDM2 knockdown. Error bars depict the standard deviations from three experiments. The P-value was calculated with Student’s t-test. (E) Knockdown of NIR and MDM2 cooperate to increase the p21 protein levels. HCT116 cultures were treated as in (D). Total protein extracts were analysed by western blotting with anti-p21 monoclonal antibody (1:1000), NIR antibody 2910 (1:2000), MDM2 antibody 3G9 (1:2000) and β-actin antibody (1:10 000). Densitometry was used to quantitate p21 signal intensity (diagram).