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. 2019 Feb 5;38(5):e99599. doi: 10.15252/embj.201899599

Figure 2. EZH2 upregulates p53 expression via binding to the IRES motif in 5′UTR .

Figure 2

  1. Biotin pull‐down assay by incubating biotin‐labeled different fragments of p53 mRNA and HOTAIR (positive control) with C4‐2 cell lysate followed by Western blot with EZH2 antibody.
  2. Biotin pull‐down assay as in (A) using unmutated and various internally deleted mutants of 5′UTR of p53 mRNA. Top, diagram of different p53 5′UTR deletion mutants.
  3. Upper, the linear map of the pRF bicistronic report plasmid. The SV40 promoter (purple box) was used to drive firefly luciferase (Fluc) and Renilla luciferase (Rluc) gene transcription. Different p53 5′UTR fragments were inserted between the Fluc and Rluc genes. Lower, at 24 h after transfection, cells were lysed and luciferase activities were measured using a dual‐luciferase kit and the ratio of Fluc/Rluc was calculated. Data shown as means ± SD (n = 3). Statistical significance was determined by two‐tailed Student's t‐test. *P < 0.01.
  4. EZH2 knockdown C4‐2 cells were transfected with the bicistronic reporter vector in combination with empty vector, Myc‐tagged EZH2 WT, or deletion mutants followed by Western blot analysis with indicated antibodies (bottom) and luciferase assay (top). Data shown as means ± SD (n = 3). *P < 0.01.
  5. C4‐2 and U2OS cell lines were transfected with non‐specific control (siC) or two independent EZH2‐specific siRNAs and harvested for Western blot analysis with indicated antibodies. ERK2 and β‐TUBULIN, loading controls.
  6. U2OS cells were transfected with control (siC) or EZH2‐specific siRNAs and treated with 200 nM of CPT followed by Western blot analysis for indicated proteins.
  7. C4‐2 cells were transfected with control (siC) or EZH2‐specific siRNAs and plasmids for empty vector, EZH2 WT, or deletion mutants followed by Western blot analysis for indicated proteins.

Source data are available online for this figure.