Fig 1: Jumonji domain‐containing 1A (JMJD1A) stimulates RUNX3 promoter through co–activation of transcription factor Ets‐1. The activities of pGL3‐P1 (A) or pGL3‐P2 (B) were measured by luciferase assays in cells transfected with increasing amounts of JMJD1A expression vector. (C) Schematic diagram depicting genomic organization of the human RUNX3 gene and the three potential transcription factor binding sites in the P1 promoter of RUNX3. Black boxes represent coding exons, whereas the other boxes represent UTR. The effect of Ets‐1 (D) or JMJD1A (E) on the activities of RUNX3 promoter P1 of wild type (WT) or the mutant (MutE) or the delete (Del) was examined by luciferase assays in HEK293 cells. (F) JMJD1A and Ets‐1 were transiently transfected into HEK293 cells as indicated, and the P1 promoter activity was estimated by luciferase assays. ***P < 0.001. (G,H) SGC‐7901 cells were subjected to ChIP assays with antibodies as indicated, followed by quantitative PCR with primers amplifying the RUNX3 promoter P1 and P2. (I) The interaction of Ets‐1 with the P1 region of RUNX3 promoter was examined by ChIP assays. SGC‐7901 cells were transfected with flag‐Ets‐1 and JMJD1A expression vector as indicated. ChIP was carried out using antibody against flag, followed by PCR with primers amplifying the RUNX3 promoter P1 (P1) or GAPDH promoter (G‐P). J, SGC‐7901 cells were transiently transfected with JMJD1A expression vector and flag‐Ets‐1 vector. Then ChIP re‐ChIP assays were carried out to examine whether JMJD1A and Ets‐1 were assembled on the same promoter (P1). Soluble chromatin was first immunoprecipitated with antibody against flag (1st IP). The complexes eluted from the 1st IP were divided into two aliquots, followed by reimmunoprecipitation with antibody against IgG or JMJD1A (2nd IP), respectively. The PCR primers were specific for amplifying the RUNX3 promoter P1 (P1) or GAPDH promoter (G‐P). (K) ChIP‐quantitative PCR (qPCR) was carried out using H3K9me1 or H3K9me2 Abs, and negative control Abs (IgG) in SGC‐7901 cells, followed by qPCR with primers amplifying the RUNX3 promoter P1 and P2 region. (L) ChIP assays were carried out with antibody against H3K9me2 in control and Ets‐1 knockdown MGC‐803 cells, followed by qPCR with primers amplifying the RUNX3 promoter P1 and P2. (M) The interaction of JMJD1A with Ets‐1 was examined by immunoprecipitation (IP) assays in SGC‐7901 cells
Fig 2: The nTCA cycle is functionally linked to transcription regulation and cellular activities. a RNA-seq analysis of differentially expressed genes in CS-depleted HepG2 cells. b Venn diagram for cross-analysis of ATAC-seq and RNA-seq in CS-deficient HepG2 cells. c Ontology analysis of genes regulated by CS depletion in HepG2 cells. d HepG2 cells were transfected with CS siRNA and/or CS∆MLS for analysis of FADD expression by qRT-PCR and western blotting. Error bars represent mean ± SD for triplicate experiments (*p < 0.05). e qChIP analysis of FADD and GAPDH promoter occupancy by CS or RUNX3 in HepG2 cells. Error bars represent mean ± SD for triplicate experiments (*p < 0.05). f HepG2 cells were transfected with CS∆MLS or/and CS siRNA followed by treatment with CPT for 48 h for western blotting analysis with antibodies against cleaved caspase-8, caspase-3, and PARP. g HepG2 cells were transfected with CS∆MLS or/and CS siRNA or FADD siRNA followed by treatment with CPT for 48 h for immunofluorescent staining with antibodies against cleaved caspase-3 (green). DAPI staining was included to visualize the nucleus (blue). Bar, 200 μm. The relative fluorescent intensity of cleaved caspase-3 was quantified by Image J software. Error bars represent mean ± SD for triplicate experiments (*p < 0.05). h HepG2 cells were transfected with CS∆MLS or/and CS siRNA or FADD siRNA followed by treatment with CPT for 48 h for Hoechst 33258 staining and analysis by fluorescence microscopy. The arrows indicate the apoptotic bodies. Bar, 200 μm. i HepG2 cells were transfected with CS∆MLS or/and CS siRNA or FADD siRNA followed by treatment with CPT for 48 h for Annexin V-FITC/PI staining and flow cytometry
Fig 3: Jumonji domain‐containing 1A (JMJD1A) expression is positively associated with runt‐related transcription factor 3 (RUNX3) expression in gastric cancer (GC) samples. (A) The expression levels of JMJD1A and RUNX3 were examined by immunohistochemistry (IHC) staining analysis in paracancerous tissue and GC tissue of tissue microarray. Scale bar, 100.8 μm. The IHC scores of JMJD1A (B) and RUNX3 (C) were quantified and plotted as graphs. (D) Spearman’s rank test was used to analyze the correlation between JMJD1A and RUNX3 relative expression in GC samples. Kaplan‐Meier survival curves of gastric cancer patients based on JMJD1A expression (E) and RUNX3 expression (F). (G) Kaplan‐Meier survival curves of gastric cancer patients based on JMJD1A expression using Kaplan‐Meier plotter database. (H) Model shows the mechanism by which JMJD1A upregulates RUNX3 by Ets‐1 in gastric cancer cells
Fig 4: Expression of RUNX3 and EZH2 in LARC tissues. (A) Low RUNX3 expression; (B) high RUNX3 expression; (C) High EZH2 expression; (D) Low EZH2 expression. Scar bar = 25 μm.
Fig 5: Kaplan-Meier analysis of the relationship between expression of RUNX3 and EZH2 and 5-year disease-free survival and overall survival in LARC patients. (A) 5-year disease-free survival of patients with high expression of RUNX3 was significantly higher compared with that of patients with low expression of RUNX3 (P < 0.05); (B) Overall survival of patients with high expression of RUNX3 was significantly higher compared with that of patients with low expression (P < 0.05). (C) 5-year disease-free survival of patients with high expression of EZH2 was significantly lower compared with that of patients with low expression of EZH2 (P < 0.05). (D) Overall survival time of patients with high expression of EZH2 was significantly lower compared with that of patients with low expression of EZH2 (P < 0.05).
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