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. 2020 Oct 25;14(12):3211–3233. doi: 10.1002/1878-0261.12819

Fig. 8.

Fig. 8

LINC01578 represses IκBβ via binding and recruiting EZH2. (A) IκBα and IκBβ mRNA levels in DLD‐1 cells transfected with LINC01578 overexpression vector were measured by qRT‐PCR. (B) IκBα and IκBβ mRNA levels in LoVo cells infected with shRNAs targeted to LINC01578 were measured by qRT‐PCR. (C) IκBβ protein level in DLD‐1 cells transfected with LINC01578 overexpression vector was measured by western blot. (D) IκBβ protein level in LoVo cells infected with shRNAs targeted to LINC01578 was measured by western blot. (E) Subcellular localization of LINC01578 was detected by qRT‐PCR in biochemically fractionated DLD‐1 cells. GAPDH, RNU1‐1, and NEAT1 were used as cytoplasmic, nucleoplasmic, and chromatinic controls, respectively. (F) The predicted interaction between LINC01578 and NFKBIB promoter. (G) ChIRP assays using LINC01578 capture probes were carried out in DLD‐1 cells. The enrichment of NFKBIB promoter and a distant NC region was determined by qPCR. (H) ChIP assays using H3K27me3 and H3K27ac antibodies were carried out in DLD‐1 cells overexpressing wide‐type or binding region‐mutated LINC01578. The enrichment of NFKBIB promoter was determined by qPCR. (I) ChIP assays using H3K27me3 and H3K27ac antibodies were carried out in LINC01578‐depleted and control LoVo cells. The enrichment of NFKBIB promoter was determined by qPCR. (J) RNA pull‐down assays were performed using in vitro‐transcribed biotinylated LINC01578. The enriched proteins were detected by western blot. GAPDH was used as NC. (K) RIP assays using EZH2 antibody were carried out in DLD‐1 cells. The enrichment of LINC01578 was determined by qRT‐PCR. GAPDH and HEIH were used as negative and positive controls, respectively. (L) ChIP assays using EZH2 antibody were carried out in LINC01578‐overexpressed and control DLD‐1 cells. The enrichment of NFKBIB promoter and a distant NC region was determined by qPCR. EZH2 protein level in LINC01578‐overexpressed and control DLD‐1 cells was determined by western blot. (M) ChIP assays using EZH2 antibody were carried out in LINC01578‐depleted and control LoVo cells. The enrichment of NFKBIB promoter and a distant NC region was determined by qPCR. EZH2 protein level in LINC01578‐depleted and control LoVo cells was determined by western blot. Data are shown as mean ± SD based on three independent experiments. **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, not significant, by Student's t‐test (A,K,L), or one‐way ANOVA followed by Dunnett's multiple comparisons test (B,G,H,I,M).