Fig 2: Dynamic enhancer–promoter interactions detected by NicE-C. (A) An example of TNF-stimulation-induced changes in E-P loops around the TNFAIP3 locus, detected by NicE-C. Snapshots of 1D chromatin tracks (ChIP-seq of H3K4me3 and H3K27ac, and DNase-seq [for references, see Methods]; open chromatin signals of NicE-C, and RNA-seq) in the example region are also shown. Red arrows pointing to e1 to e6 show the putative enhancers based on ChIP-seq, DNase-seq, and our NicE-C peaks. The region in orange represents the gene promoter. Ovals indicate examples of increased interactions associated with indicated gene promoter in TNF-treated cells compared to control cells. (B) An example of E-P loops in old mouse kidney compared to those in young mouse kidney around the Acsm3 locus (down-regulated gene in old mouse kidney), detected by NicE-C. Snapshots of 1D chromatin tracks (ChIP-seq of H3K4me3 and H3K27ac, DNase-seq, and open chromatin signals of NicE-C and RNA-seq) in this region are also shown. Red arrows pointing to e1 to e4 show the putative enhancers based on ChIP-seq, DNase-seq, and our NicE-C peaks. The region in orange represents the gene promoter. Ovals indicate examples of decreased interactions associated with indicated gene promoter in old kidney compared to young kidney.

Fig 3: PI-103 induces cell death independent of apoptosisA. RT4 cells were treated with a series of concentrations of PI-103 (PI) alone, or with pan-caspase inhibitor zVAD-fmk (zVAD) (20uM) for 24 hours. Cell viability was measured via ATP assay kit (Promega) (n = 6 per condition). Data are shown as mean ±sd (note that the error bars are not visible due to minor variations). B. RT4 cells were treated with PI (0.5uM) with or without zVAD (20uM) for the time periods indicated, and cell viability was measured via MTT assay (n = 6 per condition). Data are shown as mean ±sd. C. HeLa cells were treated with vehicle, PI (5uM) or TNF (30ng/ml)+cycloheximide (CHX, 30uM) (as a positive control) for 20 hours and analysed by immunoblot with the indicated antibodies. D. HeLa cells were transfected with control or caspase-3 siRNA for 24 hours then treated with vehicle or PI (5uM) for 48 hours. Cell death was measured with propidium iodide staining (n = 6 per condition). TNF+CHX±zVAD was used as additional controls for apoptosis inhibition. Data are shown as mean ±sd. ***: P < 0.001, *: P < 0.05. Immunoblot was used to verify knockdown efficiency. E. HeLa cells were transfected with control or caspase-8 siRNA for 24 hours then treated with vehicle or PI (5uM) for 20 hours. Cell death was measured with propidium iodide staining (n = 6 per condition). Data are shown as mean ±sd. **: P < 0.01. Immunoblot was used to verify knockdown efficiency. F. HeLa cells were transfected with control or caspase-9 siRNA for 24 hours then treated with vehicle or PI (5uM) for 48 hours. Cell death was measured with propidium iodide staining (n = 6 per condition). Data are shown as mean ±sd. Immunoblot was used to verify knockdown efficiency. G. WT MEF or Bax/Bak DKO MEFs were treated with vehicle, PI (3uM), or STS (1uM) for 24 hours. Cell viability was measured via ATP assay kit (Promega) (n = 12 per condition). Data are shown as mean ±sd.

Fig 4: TNF pathway activation mediated p38 MAPK-eIF4E axis signaling and B7-H3 upregulation. (A) RNA-seq heatmap represents the differential gene expressions in SP20H or F118A knockout cells incubated for various periods of time. The gene expression value in heatmap was normalized by log2-fold change in each row. (B) Bar plot shows the most significant enriched GO terms and KEGG pathways in D7_SP20H-knockout cells. (C and D) GSEA plots of indicated signature genes enriched in long-term SP20H depleted cells (D7_S). (E–F) SK-OV-3 and DU145 cells treated or untreated with different concentrations of TNF-a were analyzed for surface B7-H3 protein expression by flow cytometric analysis and detected for total protein expression of B7-H3, phospho-p38, and phospho-eIF4E by immunoblotting. At least two independent experiments were performed with similar results; representative data are shown. D3/D7: day3, day7; F, F118A; GO, Gene Ontology; GSEA, gene set enrichment analysis; S, SP20H.