Fig. 2.

LINC01578 is upregulated by NF‐κB and YY1. (A) The predicted NF‐κB and YY1 binding sites on LINC01578 promoter. NF‐κB and YY1 bind sites were at −438 and −4 positions relative to the transcription start site, respectively. (B) ChIP assays were performed in DLD‐1 cells to measure the binding of NF‐κB and YY1 on LINC01578 promoter. A distant region without NF‐κB and YY1 binding sites was used as NC (P3). (C) Luciferase reporter assays for DLD‐1 cells transfected with luciferase reporter plasmids containing LINC01578 promoter and treated with PBS or 10 ng·mL⁻¹ TNF‐α for 24 h. Nuclear p65 levels of DLD‐1 cell treatment with PBS or 10 ng·mL⁻¹ TNF‐α for 24 h were detected by western blot. (D) Luciferase reporter assays for DLD‐1 cells transfected with luciferase reporter plasmids containing LINC01578 promoter and treated with DMSO or 5 µm JSH‐23 for 24 h. Nuclear p65 levels of DLD‐1 cell treatment with DMSO or 5 µm JSH‐23 for 24 h were detected by western blot. (E) Luciferase reporter assays for DLD‐1 cells cotransfected with luciferase reporter plasmids containing LINC01578 promoter and p65 overexpression vector. p65 overexpression efficiencies were detected by western blot. (F) Luciferase reporter assays for DLD‐1 cells cotransfected with luciferase reporter plasmids containing LINC01578 promoter and siRNAs against p65. p65 knockdown efficiencies were detected by western blot. (G) Luciferase reporter assays for DLD‐1 cells cotransfected with luciferase reporter plasmids containing LINC01578 promoter and YY1 overexpression vector. YY1 overexpression efficiencies were detected by western blot. (H) Luciferase reporter assays for DLD‐1 cells cotransfected with luciferase reporter plasmids containing LINC01578 promoter and siRNAs against YY1. YY1 knockdown efficiencies were detected by western blot. (I) LINC01578 expression in DLD‐1 cells treated with PBS or 10 ng·mL⁻¹ TNF‐α for 24 h. (J) LINC01578 expression in DLD‐1 cells treated with DMSO or 5 µm JSH‐23 for 24 h. (K) LINC01578 expression in DLD‐1 cells transfected with p65 overexpression vector. (L) LINC01578 expression in DLD‐1 cells transfected with siRNAs against p65. (M) LINC01578 expression in DLD‐1 cells transfected with YY1 overexpression vector. (N) LINC01578 expression in DLD‐1 cells transfected with siRNAs against YY1. Data are shown as mean ± SD based on three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant, by one‐way ANOVA followed by Dunnett's multiple comparisons test (B) or Student's t‐test (C‐N).