Fig 2: Western blot analysis of NF-κB p50 and p65 in cytoplasmic and nuclear protein extract of macrophages. Proteins were quantified by Lowry methods and equal amount of protein (40 μg/lane) was subjected to SDS–PAGE followed by western blotting using NF-κB p50 and p65 specific monoclonal antibodies. The expression of NF-κB p50 and p65 in cytoplasmic and nuclear proteins was observed by gel documentation system and the bands’ intensity was quantified by densitometry by Quantity One software. The NF-κB p50 and p65 expression in cytoplasmic protein of antagomir treated macrophages was significantly (p < 0.001) decreased compared to untreated macrophages (A). Further, in nuclear protein, the NF-κB p50 and p65 expression in antagomir treated macrophages was significantly (p < 0.001) increased (B). Antagomir scramble did not affect the either cytoplasmic or nuclear protein expression.

Fig 3: LINC01578 forms a positive feedback loop with NF‐κB/YY1. (A) YY1 mRNA and protein levels in DLD‐1 cells transfected with LINC01578 overexpression vector were determined by qRT‐PCR and western blot. (B) YY1 mRNA and protein levels in LoVo cells infected with shRNAs targeted to LINC01578 were determined by qRT‐PCR and western blot. (C) YY1 mRNA and protein levels in DLD‐1 cells transfected with LINC01578 overexpression vector and treated with 5 µm JSH‐23 were determined by qRT‐PCR and western blot. (D) YY1 mRNA and protein levels in LoVo cells infected with shRNAs targeted to LINC01578 and treated with 5 µm JSH‐23 were determined by qRT‐PCR and western blot. (E) A schematic model of positive feedback loop between LINC01578 and NF‐κB/YY1. (F) Luciferase reporter assays for LINC01578‐overexpressed and control DLD‐1 cells transfected with luciferase reporter plasmids containing LINC01578 promoter. (G) Luciferase reporter assays for LINC01578‐depleted and control LoVo cells transfected with luciferase reporter plasmids containing LINC01578 promoter. (H) ChIP assays using p50 and p65 antibodies were carried out in LINC01578‐overexpressed and control DLD‐1 cells. The enrichment of LINC01578 promoter was determined by qPCR. (I) ChIP assays using p50 and p65 antibodies were carried out in LINC01578‐depleted and control LoVo cells. The enrichment of LINC01578 promoter was determined by qPCR. (J) ChIP assays using YY1 antibody were carried out in LINC01578‐overexpressed and control DLD‐1 cells. The enrichment of LINC01578 promoter was determined by qPCR. (K) ChIP assays using YY1 antibody were carried out in LINC01578‐depleted and control LoVo cells. The enrichment of LINC01578 promoter was determined by qPCR. Data are shown as mean ± SD based on three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, ns, not significant, by Student's t‐test (A,C,F,H,J), or one‐way ANOVA followed by Dunnett's multiple comparisons test (B,D,G,I,K).

Fig 4: LINC01578 activates NF‐κB signaling. (A) p50 IHC staining of liver metastatic tumors derived from LINC01578‐overexpressed and control DLD‐1 cells. Scale bars, 50 µm. (B) p50 IHC staining of liver metastatic tumors derived from LINC01578‐depleted and control LoVo cells. Scale bars, 50 µm. (C) Nuclear p50 and p65 levels in LINC01578‐overexpressed and control DLD‐1 cells were detected by western blot. (D) Nuclear p50 and p65 levels in LINC01578‐depleted and control LoVo cells were detected by western blot. (E) Firefly luciferase reporters containing NF‐κB binding sites (pNFκB‐luc) were transfected into LINC01578‐overexpressed and control DLD‐1 cells. Then, luciferase reporter assays were performed to determine NF‐κB transcriptional activity. (F) Firefly luciferase reporters containing NF‐κB binding sites (pNFκB‐luc) were transfected into LINC01578‐depleted and control LoVo cells. Then, luciferase reporter assays were performed to determine NF‐κB transcriptional activity. (G) p50 and p65 activation in nuclear extracts from LINC01578‐overexpressed and control DLD‐1 cells was determined by NF‐κB p50 Transcription Factor Assay Kit and NF‐κB p65 Transcription Factor Assay Kit, respectively. (H) p50 and p65 activation in nuclear extracts from LINC01578‐depleted and control LoVo cells was determined by NF‐κB p50 Transcription Factor Assay Kit and NF‐κB p65 Transcription Factor Assay Kit, respectively. Data are shown as mean ± SD based on n = 6 mice in each group (A,B) or three independent experiments (C–H). *P < 0.05, **P < 0.01 by the Mann–Whitney test (A), Kruskal–Wallis test followed by Dunn's multiple comparisons test (B), or one‐way ANOVA followed by Dunnett's multiple comparisons test (E–H).