Fig 2: LINC01578 represses I?Bß via binding and recruiting EZH2. (A) I?Ba and I?Bß mRNA levels in DLD-1 cells transfected with LINC01578 overexpression vector were measured by qRT-PCR. (B) I?Ba and I?Bß mRNA levels in LoVo cells infected with shRNAs targeted to LINC01578 were measured by qRT-PCR. (C) I?Bß protein level in DLD-1 cells transfected with LINC01578 overexpression vector was measured by western blot. (D) I?Bß 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).

Fig 3: Role of KIT-regulated NFKBIB in GIST cell function. a, b GIST48 and GIST430 cells were transfected with 150 nM siRNA targeting NFKBIB or a scrambled control (SC) for 18 h. a The transfected cells were suspended, and equal numbers of cells were seeded into 24-well plates. After 6 h, the cells were attached and examined as the day 0 (D0) control, and the other cells were examined at the indicated times using a relative cell viability assay. b The cells were lysed and analyzed by immunoblotting. Actin served as an internal control. c–g Cells were transfected with siRNA targeting NFKBIB or a scrambled control for 72 h. The cells were analyzed by Annexin V staining (c) or immunoblotting against PARP1 (d). e The transfected cells were separated into cytoplasmic and nuclear fractions and analyzed by immunoblotting. LMNB1 and GAPDH were used as nuclear and cytoplasmic markers, respectively. f Nuclear proteins were used to analyze the RELA transcriptional activity. g Chromatin from the transfected cells was cross-linked, sheared, immunoprecipitated using an anti-RELA antibody, and amplified by PCR. Chromatin that was sheared but not immunoprecipitated was used as an input control. All experiments were repeated at least three times. h Representative GIST tissue samples were analyzed by immunostaining against p-KIT (KITY703; green) and NFKBIB (red) and visualized by confocal microscopy. Using photomicrographs, GISTs were classified as low-risk, moderate-risk, or high-risk according to NCCN consensus criteria based on increasing mitoses. i The scatter plot showed the correlations between 96 GIST tissues with various risk levels and the H-scores of the immunoexpression levels of nuclear KITY703 and NFKBIB. The box plot showed the associations between the nuclear KITY703 (j) and NFKBIB (k) immunoexpression levels and the H-scores for tumor grade based on the NCCN risk level according to the Kruskal–Wallis test. The middle line demonstrated the median, the box illustrated the interquartile range, and the whiskers indicated the extreme data points >1.5x the interquartile range from the box. *p < 0.05

Fig 4: Schematic of the KIT-NFKBIB-RELA autoactivation loop in mutant KIT-expressing GIST cells