Fig. 1.

RBEL1A negatively regulates expression of p53 and its target genes p21 and PUMA. (A) Increased RBEL1A expression is associated with reduced p53 protein levels in RBEL1A-overexpressing cells. Western blot analyses showing the expressions of p53, Puma, p21, MDM2, Erk1/2, PI3K, cyclin D₃, HA–RBEL1A in cells stably overexpressing RBEL1A or empty vector. β-actin served as loading control. All results shown in the left panel and those in the top right panel were from the same membrane; images in the right bottom panel were from the duplicate membrane using the same lysates. (B) RBEL1A knockdown increases p53 levels and expression of its target genes. MCF-7 cells were infected (MOI = 0.3) with lentivirus carrying either scrambled shRNA or RBEL1A-specific shRNA. Ten days post-infection, cells were harvested and western blot analyses were performed using the indicated antibodies. β-actin was also detected on the same membrane as a loading control. (C) RBEL1A knockdown increases p53 protein half-life. MCF-7 cells infected with lentivirus-mediated RBEL1A shRNA or with the scramble shRNA (10 days) were treated with cycloheximide (50 µg/ml) and harvested at various times as indicated. The RBEL1A, p53 and β-actin protein signals were detected by western blotting on the same membrane. (D) The values of relative intensity were obtained based on the densitometry measurements of p53 and β-actin levels on the western blotting shown in C. (E) Results of real-time qPCR of p53, p21 and Puma transcripts in RBEL1A-overexpressing and control cells. Real-time qPCR assays were performed as described in the Materials and Methods. The data are from three independent experiments in which each sample was tested in triplicate. **P<0.01 as determined by a t-test. (F) RBEL1A-mediated p53 downregulation is blocked by the proteasome inhibitors MG132 and Nutlin-3. Cells stably overexpressing RBEL1A or empty vector (control) were either not treated or treated with proteasome inhibitor MG132 (10 µM) or Nutlin-3 (10 µM) for 5 hours. The expression levels of endogenous p53 and β-actin were detected on the same membrane. The numbers below the p53 panel indicate the relative intensity of p53 expression.