Figure 4
From: The orphan nuclear receptor NR4A2 is part of a p53–microRNA-34 network
NR4A2 overexpression suppresses p53 activation.
HCT116 cells were transduced for 16 h with lentivirus expressing empty vector (EV) or 3xFlag-NR4A2 (NR4A2). The cell medium was then changed and the cells remained in culture for a total of 48 h. The cells were then reseeded and treated with vehicle (DMSO) or Nutlin-3a (5 or 10 μM) for 24 or 48 h. Expression of NR4A2 (a) mir-34a (b) CDKN1A (c) and TP53 (d) was determined using TaqMan qPCR probes (normalized to GAPDH). The value for DMSO-treated EV at each time point was set as 1. The statistical significance of the difference between the results with EV and NR4A2 for each treatment was determined for each time point by using a two-way ANOVA with Sidak’s multiple comparisons test. ####P ≤ 0.0001; ###P ≤ 0.001; ##P ≤ 0.01; #P ≤ 0.05. (e) Whole-cell lysates from HCT116TP53−/− (KO) and HCT116 wild-type (WT) cells transduced with lentivirus expressing EV or 3xFlag-NR4A2 (F-NR4A2) were assessed for expression of Flag (indicating NR4A2), p53 and Gapdh protein by performing SDS-PAGE gel electrophoresis. Relative p53 expression was determined using Odyssey Image Studio to calculate the ratio of p53 to Gapdh protein band density (displayed at the bottom of the gel). The ratio for EV in WT cells was set as 1.0.
