Fig 2: Olaparib treatment enhances the interaction of MDM2 with RPL5 and RPL11. (A) HCT116 p53+/+ cells were transfected with Flag-L5 and treated with or without 20 µM Olaparib, and subject to the immunofluorescence assay. (B) The experiment was performed as the same as (A), except that Flag-L11 was introduced into cells. (C) Cal51 cells were treated with or without 20 µM Olaparib for 24 h and harvested for co-IP-IB analysis. RPL5 was co-immunoprecipitated with MDM2 using an anti-MDM2 antibody. (D) The experiment was performed as the same as (C), except that RPL11 was co-immunoprecipitated with MDM2 using an anti-MDM2 antibody.

Fig 3: Lysine residues at positions 152 and 154 in ORF50 are critical for MDM2-mediated degradation.(A) Schematic diagram of ORF50 and ORF50 lysine mutants. The positions of 25 lysines in ORF50 are shown in the diagram (circles). Black circles represent the substitutions of lysine (K) with arginine (R) in F-ORF50. (B) Immunoblot analysis of lysine substitution mutants of ORF50. Cell lysates of 293T cells that were transfected with the indicated expression plasmids were probed with anti-FLAG antibody. (C) Effects of single- or double-lysine mutations from K124 to K243 (the middle 8 lysine clusters) on ORF50 abundance. (D) Susceptibility of ORF50 mutants to MDM2-mediated degradation. The expression plasmids encoding ORF50 mutants were cotransfected with an MDM2 expressing plasmid or control vector into 293T cells. The expression of these ORF50 mutants in the presence or absence of exogenous MDM2 was determined by immunoblotting with anti-FLAG antibody. (E) Relative protein levels of ORF50 mutants in the presence or absence of overexpressed MDM2. The bar graph summarizes densitometry data from three independent experiments. Error bars: standard deviation.

Fig 4: Working model of Olaparib activation of p53 via nucleolar stress. Under the normal condition, RPs and rRNAs work together for pre-ribosome assembly in the nucleolus, while MDM2 binds to p53 and maintains a relatively low level of p53 in cells (left panel). Olaparib treatment inhibits pre-rRNA biosynthesis, thus leading to nucleolar stress. Many RPs, such as RPL5 and RPL11, are released to the nucleoplasm to interact with MDM2 resulting in p53 stabilization and activation, and consequent p53 target gene upregulation.

Fig 5: Sp1 is involved in the ORF50-mediated transcriptional repression of the P1 promoter of the MDM2 gene. (A) Point mutational analysis of the P1(–103/–63) promoter element (or referred to as NRE-1). The diagram shows the nucleotide sequence of NRE-1 and its mutated elements (top panel). Notably, there are three Sp1-binding sites predicted in the NRE-1 element. The responsiveness of the reporter constructs containing the NRE-1 element or its mutants (M1, M2, and M3) to ORF50 was assayed in 293T cells (bottom panel). *, p < 0.05, compared to pE4-luc; #, p < 0.05, compared to the NRE-1 group (n = 3). (B) EMSA analysis of the NRE-1 element using total protein extracts of 293T cells transfected with the empty vector or the ORF50 expression plasmid. Four major protein/DNA complexes (C1, C2, C3, and C4) were detected in the EMSA. Cold competitors shown in panel (A) were added at a concentration of a 50-fold molar excess relative to the labeled probe. (C) Antibody supershifting or blocking test in EMSA. Specific antibodies to Sp1, Sp3, KLF4, or actin were used for EMSA supershift or blockade assay. (D) Direct binding of Sp1 or the Sp1 DNA-binding domain (Sp1-DBD) to the NRE-1 element. EMSA was performed using total protein extracts of 293T cells transfected with the empty vector or the plasmid expressing the full-length Sp1 or Sp1-DBD. (E) Point mutational analysis of the P1(–39/+1) promoter element (or referred to as NRE-2). The nucleotide sequences of NRE-2 and its mutated elements are shown in the diagram (top panel). Note that there are two Sp1-binding sites predicted in the NRE-2 element. The responsiveness of the indicated reporter constructs to ORF50 were determined in 293T cells (bottom panel). *, p < 0.05, compared to pE4-luc; #, p < 0.05, compared to the NRE-2 group. (F) Direct binding of Sp1 to the NRE-2 element. EMSA was performed using total protein extracts of 293T cells transfected with the empty vector or the Sp1 expression plasmid. Cold competitors shown in panel (E) were added at a concentration of a 30-or 60-fold molar excess relative to the labeled probe. (G) Effects of Sp1, Sp3, p53, or ORF50 on the expression of MDM2 in 293T cells. 293T cells were transfected with the indicated expression plasmids, and the protein expression of MDM2 in these transfected cells was analyzed by immunoblotting. (H) Effect of ORF50 on the Sp1-mediated MDM2 upregulation in 293T cells. Increasing amounts of the Sp1 expression plasmid were cotransfected with the empty vector or the ORF50 expression plasmid into 293T cells for 30 h. The expression levels of MDM2, Sp1, and ORF50 in these transfected cells were determined by Western blot analysis. (I) Proposed model for the repressive effect of ORF50 on Sp1-mediated activation of the MDM2 P1 promoter.