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. 2021 Aug 30;22(10):e52537. doi: 10.15252/embr.202152537

Figure 3. SQLE is a transcriptional target of p53.

Figure 3

  1. Schematic representation of human SQLE genomic structure. Shown are the exon/intron organization and two potential p53 response elements (RE1 and RE2) and the corresponding mutant response elements.
  2. p53+/+ and p53 −/− HCT116 cells were analyzed by chromatin immunoprecipitation (ChIP) assay using normal IgG and anti‐p53 antibody as indicated.
  3. p53+/+ HCT116 cells transfected with control siRNA or SREBP2 siRNA for 72 h were analyzed by ChIP assay with the indicated antibodies.
  4. Control and SREBP2 knockout MCF‐7 cells using sgRNA CRISPR/Cas9 were analyzed by ChIP assay using anti‐p53 or normal mouse IgG antibodies.
  5. The electrophoretic mobility shift assay (EMSA) of SQLE‐RE2 or RE2 mut in the presence or absence of p53 antibodies as indicated. Blue box (shift band) means the binding between nuclear extracts and RE2, red box (super‐shift band) means p53 as the protein presented in the EMSA band.
  6. Luciferase reporter constructs containing RE1, RE2, RE1mut, or RE2mut were transfected into p53 −/− HCT116 cells together with control (PRK5‐flag vector) or p53 expression vector (PRK5‐flag p53) for 48 h. Renilla vector pRL‐CMV was used as a transfection internal control. Relative levels of luciferase are shown. Data represent three independent experiments. Protein expression is shown.
  7. Luciferase reporter constructs containing RE1, RE2, RE1mut, or RE2mut were transfected into HEK293T cells together with control (PRK5‐flag vector) or p53 expression vector (PRK5‐flag p53) for 48 h. Renilla vector pRL‐CMV was used as a transfection internal control. Relative levels of luciferase are shown. Protein expression is shown.
  8. Luciferase reporter constructs containing RE2 were transfected into HEK293T cells together with control, wild‐type p53, or p53 R175H expression vector for 48 h. Renilla vector pRL‐CMV was used as a transfection internal control. Relative levels of luciferase are shown.
  9. Luciferase reporter assay with indicated response element constructs in human HEK293T cells co‐transfected with or without p53 expression vector.
  10. Luciferase reporter constructs containing RE2 were transfected into HEK293T cells treated with or without 20 μm PFTα for 24 h. Renilla vector pRL‐CMV was used as a transfection internal control. Relative levels of luciferase are shown.

Data information: In (B, C, D, F, G, H, I, J), bars represent mean ± s.d., *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; n = 3 biologically independent samples; statistical significance was determined by two‐tailed unpaired t‐test.