Journal: CNS Neuroscience & Therapeutics

Article Title: Alpha‐lipoic acid downregulates TRPV1 receptor via NF‐κB and attenuates neuropathic pain in rats with diabetes

doi: 10.1111/cns.13303

Figure Lengend Snippet: ALA inhibited TRPV1 expression via suppressing NF‐κB. A, Western blots for NF‐κB in DRGs innervating the hindpaw from control and STZ‐induced diabetic rats. Bar graph involved mean density relative of β‐actin and NF‐κB from control and STZ‐induced diabetic rats. STZ injection significantly enhanced expression of NF‐κB (N = 4 for each group; * P < .05, compared with CNT, two‐sample t test) in L4‐L6 DRGs. B, Western blots for TRPV1 in L4‐L6 DRGs from diabetic rats treated with p65 siRNA lentivirus and NC siRNA lentivirus, respectively. Graph showed mean density relative to GAPDH for TRPV1. The lentiviruses were intrathecally injected into rats. The expression of TRPV1 significantly reduced after p65 siRNA lentiviruses treatment compared with NC siRNA group (N = 4 for each group, * P < .05, compared with NC siR, two‐sample t test). C, Western blots for NF‐κB in L4‐L6 DRGs from diabetic rats treated with NS and ALA, respectively. Graph showed mean density relative to GAPDH for NF‐κB. ALA treatment strongly reduced the expression of NF‐κB (N = 4 for NS group, N = 3 for ALA group, ** P < .01, compared with NS, two‐sample t test)

Article Snippet: Anti‐TRPV1 (#ACC‐030, Alomone, 1:200), anti‐p65 (#6956, Cell Signaling Technology, 1:1000), and corresponding horseradish peroxidase‐conjugated secondary anti‐rabbit and anti‐mouse antibodies at dilutions of 1:2000 and 1:2000 were used to probe the proteins, respectively.

Techniques: Expressing, Western Blot, Injection

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: DHX9 contributes to the malignant phenotypes of colorectal cancer via activating NF-κB signaling pathway

doi: 10.1007/s00018-021-04013-3

Figure Lengend Snippet: DHX9 mediates the activation of NF-κB signal pathway in colorectal cancer cells. A Heatmap showed the alteration of gene expression (≥ twofold) obtained from RNA-Seq of vector-transfected and DHX9-silenced HCT116 cells. B Scatter plot displayed fold changes of gene expression (≥ twofold) in DHX9-silenced HCT116 cells compared with vector-transfected cells. C GO analysis of down-regulated genes (≥ twofold) in DHX9-silenced HCT116 cells compared with vector-transfected cells. D-E GSEA revealed that gene sets of cell motility D and NF-κB signaling E were enriched in DHX9-depleted HCT116 cells. F Proteins levels of IKBα, p65 and phosphorylated IKBα and p65 were evaluated by Western blotting in DHX9-overexpressed or -depleted HCT116 cells. G-H Cytosolic and nuclear fractionations were evaluated by Western blotting in DHX9-overexpressed HCT116 cells with or without p65 knockdown G or in DHX9-silenced HCT116 cells with or without LPS (1 μg/ml) pretreatment for 15 min H. I DHX9-overexpressed or -depleted HCT116 cells were subjected to immunofluorescence analysis of p65 localization (green). Vector-transfected (pLVX) HCT116 cells pretreated with LPS (1 μg/ml) for 15 min were served as a positive control. DAPI (blue) was applied to stain the nuclear. Scale bar, 20 μm. J HCT116 cells with stably expressing pLVX, pLVX-DHX9 or shRNAs against DHX9 were co-transfected with 0.5 μg NF-κB-TATA-Luc reporter plasmid and 10 ng Renilla luciferase reporter. 36 h after transfection, pLVX-HCT116 cells were treated with LPS (200 ng/mL) for 8 h. The luciferase activity of cells was measured. The values of firefly luciferase activity were normalized to Renilla luciferase activity. Fold activation were represented as mean ± SEM of three independent transfections. ***P < 0.001, one-way ANOVA with post hoc intergroup comparison by Tukey's test. K RT-qPCR experiment analyzed NF-κB-target genes including CXCL8, encoding IL-8; CCND1, encoding Cyclin D1; BIRC5, encoding Survivin; SNAI1, encoding Snail, in DHX9-overexpressed or -depleted HCT116 cells. Data were from three independent experiments and represented as mean ± SEM. ns, not significant; ***P < 0.001, one-way ANOVA with post hoc intergroup comparison by Tukey's test. L Western blotting analysis verified the protein levels of candidate NF-κB-target genes in DHX9-overexpressed or -depleted cells

Article Snippet: The cell lysates were sonicated to shear DNA to sizes of 200–500 bp and centrifuged at 12,000 g at 4 °C for 10 min. To preclear the chromatin, 60 µL of protein G agarose was added to each tube before immunoprecipitated with 5 µ g anti-p65 (Cat# 6956S, Cell Signaling Technology), anti-DHX9 (Cat# A300-855A, Invitrogen), anti-RNA pol II (Cat# A300-653A) or normal rabbit IgG (Cat# 2729S, Cell Signaling Technology) overnight at 4 °C with rotation.

Techniques: Activation Assay, Expressing, RNA Sequencing Assay, Plasmid Preparation, Transfection, Western Blot, Immunofluorescence, Positive Control, Staining, Stable Transfection, Luciferase, Activity Assay, Comparison, Quantitative RT-PCR

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: DHX9 contributes to the malignant phenotypes of colorectal cancer via activating NF-κB signaling pathway

doi: 10.1007/s00018-021-04013-3

Figure Lengend Snippet: DHX9 interacts with p65 and RNA Pol II to active NF-κB-mediated transcription in colorectal cancer cells. A-B Lysates of DHX9-overexpressed or -depleted HCT116 cells were subjected to immunoprecipitation with the anti-DHX9 or anti-IgG antibody, and then incubated with the indicated antibodies using Western blotting. C–D Lysates of DHX9-overexpressed or -depleted HCT116 cells were subjected to immunoprecipitation with the anti-p65 or anti-IgG antibody and subjected to Western blotting. E–G Stable HCT116 cells expressing WT DHX9 and K417R DHX9 were harvested and lysates were used to immunoprecipitation with the anti-DHX9 or anti-p65 or anti-IgG antibody, and then subjected to Western blotting. K417R indicates a negative mutant of DHX9, in which Lys 417 is substituted to Arg that abolishes the ATP-dependent helicase activity. H Stable HCT116 cells expressing WT DHX9 or K417R DHX9 were co-transfected with NF-κB-TATA-Luc reporter plasmid and Renilla luciferase reporter. The luciferase activity of cells was measured after 48-h transfection. The values of firefly luciferase activity were normalized to Renilla luciferase activity. Fold activation were represented as mean ± SEM of three independent transfections. ***P < 0.001, one-way ANOVA with post hoc intergroup comparison by Tukey's test

Article Snippet: The cell lysates were sonicated to shear DNA to sizes of 200–500 bp and centrifuged at 12,000 g at 4 °C for 10 min. To preclear the chromatin, 60 µL of protein G agarose was added to each tube before immunoprecipitated with 5 µ g anti-p65 (Cat# 6956S, Cell Signaling Technology), anti-DHX9 (Cat# A300-855A, Invitrogen), anti-RNA pol II (Cat# A300-653A) or normal rabbit IgG (Cat# 2729S, Cell Signaling Technology) overnight at 4 °C with rotation.

Techniques: Immunoprecipitation, Incubation, Western Blot, Expressing, Mutagenesis, Activity Assay, Transfection, Plasmid Preparation, Luciferase, Activation Assay, Comparison

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: DHX9 contributes to the malignant phenotypes of colorectal cancer via activating NF-κB signaling pathway

doi: 10.1007/s00018-021-04013-3

Figure Lengend Snippet: DHX9 is required for p65 and RNA Pol II recruitment to the promoters of NF-κB-dependent genes. A–C ChIP-qPCR analysis for DHX9, p65 or RNA Pol II occupancy at the promoter of BIRC5 A, SNAI1 B or ACTB C in HCT116 cells stably expressed DHX9 cDNA or shDDR1 constructs. All data in bar graphs were shown as mean ± SEM from three independent experiments and analyzed by one-way ANOVA, post hoc intergroup comparisons, Tukey’s test. D A proposed working model of DHX9 in orchestrating the malignant phenotypes of CRC. On one hand, DHX9 enhances p65 phosphorylation, promotes p65 nuclear translocation to facilitate NF-κB-mediated transcriptional activity. On the other hand, DHX9 interacts with p65 and RNA Pol II and is necessary to recruit p65 and RNA Pol II to the NF-κB-dependent promoters to activate downstream gene transcription, including CXCL8, CCND1, BIRC5 and SNAI1. Enhanced CXCL8, Cyclin D1 and Survivin expression contributes to lower apoptosis and promote proliferation in CRC cells; the strengthened expression of Snail increases the capability of migration and invasion, and ultimately facilities liver colonization and metastasis in CRC

Article Snippet: The cell lysates were sonicated to shear DNA to sizes of 200–500 bp and centrifuged at 12,000 g at 4 °C for 10 min. To preclear the chromatin, 60 µL of protein G agarose was added to each tube before immunoprecipitated with 5 µ g anti-p65 (Cat# 6956S, Cell Signaling Technology), anti-DHX9 (Cat# A300-855A, Invitrogen), anti-RNA pol II (Cat# A300-653A) or normal rabbit IgG (Cat# 2729S, Cell Signaling Technology) overnight at 4 °C with rotation.

Techniques: Stable Transfection, Construct, Translocation Assay, Activity Assay, Expressing, Migration

Journal: Diabetologia

Article Title: RELA governs a network of islet-specific metabolic genes necessary for beta cell function

doi: 10.1007/s00125-023-05931-6

Figure Lengend Snippet: Beta cell-specific p65 knockout dampens islet inflammation but impairs insulin secretion in response to a glucose challenge. ( a ) Real-time quantitative PCR analysis of Rela mRNA in islets isolated from littermate mice wild-type for p65 (p65fl/fl) or with beta cell-specific knockout of p65 (βp65KO). ( b ) Immunoblot of lysates from islets isolated from p65fl/fl or βp65KO littermate mice and stimulated with recombinant TNF for the times indicated. Proteins (kDa) assessed included components of the canonical NF-κB signalling pathway, phosphorylated and total JNK (p-JNK and T-JNK, respectively) and a β-actin loading control. Representative of three independent experiments. ( c ) Cumulative densitometry (relative units) of immunoblots represented in ( b ), illustrated as heat maps. Data compared against wild-type floxed (fl) 0 h sample in each blot. The flow diagram of major signalling nodes illustrates the position of each signalling event with respect to the transcription factor Rela /p65 (red). ( d ) Insulin-stained pancreatic sections (scale bar: 100 µm) from 8 week old female mice of the indicated genotypes. ( e ) Real-time quantitative PCR analysis of inflammatory mRNAs in islets isolated from littermate p65fl/fl or βp65KO mice and stimulated with TNF for the times indicated. Data are fold change relative to no TNF stimulation. ( f ) Weight and ( g ) fasting blood glucose levels of mice with or without beta cell-specific knockout of p65. F, female; M, male. ( h , i ) Blood glucose levels were monitored following an ( h ) i.p. GTT (2 g/kg glucose) (p65fl/fl, n =17; βp65KO, n =17) or ( i ) i.v. GTT (1 g/kg glucose) (p65fl/fl, n =9; βp65KO, n =9) in female mice. ( j ) Blood insulin levels (pmol/l) were measured following i.v. injection in ( i ) (p65fl/fl, n =9; βp65KO, n =9). ( k ) Beta cell mass and ( l ) in vitro GSIS assay (20 mmol/l) in islets isolated from mice with or without beta cell-specific knockout of p65. ( m ) Blood glucose levels before (day 0) and following minimal mass transplant of islets from mice with or without beta cell-specific knockout of p65 under the kidney capsule of wild-type syngeneic diabetic recipients (βp65KO, n =7; p65fl/fl, n =4). ( n ) Insulin-stained sections of islet graft 30 days post transplant (scale bar: 100 µm). Statistical analysis was performed using Student’s t tests ( a , e–g , k , l ) or AUCs ( h–j , m ). Data are means ± SEM. * p <0.05, ** p <0.01, *** p <0.001. BGL, blood glucose level; TAB, TAK1-binding protein; TAK1, TGF-β-activated kinase 1

Article Snippet: Membranes were incubated using standard techniques, diluents and buffers with anti-A20 (56305/D13H3), anti-IκBα (9242), anti-phospho-IκBα (2859/I4D4), anti-IKKγ (2585), anti-JNK (9252), anti-phospho-JNK (9255), anti-p65 (6956/L8F6) all sourced from Cell Signaling Technology, anti-mCherry (ab183628) (Abcam) or anti-β-actin (AC-15) (Sigma-Aldrich) antibodies, followed by labelling with the HRP-conjugated secondary antibody goat-anti-mouse IgG Fc (Pierce Antibodies) or donkey-anti-rabbit IgG (GE Life Sciences).

Techniques: Knock-Out, Real-time Polymerase Chain Reaction, Isolation, Western Blot, Recombinant, Control, Staining, Injection, In Vitro, Binding Assay

Journal: Diabetologia

Article Title: RELA governs a network of islet-specific metabolic genes necessary for beta cell function

doi: 10.1007/s00125-023-05931-6

Figure Lengend Snippet: Islet-derived inflammatory genes harbour p65 binding sites. ( a–c ) Representative plots of p65 ChIP-seq and corresponding accessibility signal enrichment at the ( a ) CXCL10 , ( b ) ICAM1 and ( c ) TNFAIP3 locuses. ChIP-seq and ATAC-seq peaks are shown underneath the respective signal coverage tracks. ( d ) p65 ChIP-seq enrichment, epigenomic annotations and high-confidence pcHi-C interactions from islet samples encompassing the CXCL1 locus. p65 ChIP-seq data were obtained from ENCODE. ATAC-seq data were obtained from Bysani et al . All browser views were generated using the WashU Epigenome Browser. Only the significant pcHi-C loops within the broadcast region are shown. ChIP, chromatin immunoprecipitation

Article Snippet: Membranes were incubated using standard techniques, diluents and buffers with anti-A20 (56305/D13H3), anti-IκBα (9242), anti-phospho-IκBα (2859/I4D4), anti-IKKγ (2585), anti-JNK (9252), anti-phospho-JNK (9255), anti-p65 (6956/L8F6) all sourced from Cell Signaling Technology, anti-mCherry (ab183628) (Abcam) or anti-β-actin (AC-15) (Sigma-Aldrich) antibodies, followed by labelling with the HRP-conjugated secondary antibody goat-anti-mouse IgG Fc (Pierce Antibodies) or donkey-anti-rabbit IgG (GE Life Sciences).

Techniques: Derivative Assay, Binding Assay, ChIP-sequencing, Generated, Chromatin Immunoprecipitation

Journal: Diabetologia

Article Title: RELA governs a network of islet-specific metabolic genes necessary for beta cell function

doi: 10.1007/s00125-023-05931-6

Figure Lengend Snippet: Human islets exhibit a large network of accessible p65 binding sites linked to genes governing metabolism. ( a ) Venn diagram showing the enrichment of islet-accessible p65 footprints in the islet enhancer hubs. p65 footprints were identified from islet ATAC-seq samples ( n =8 healthy donors) available from Bysani et al and intersected with islet enhancer hubs reported by Miguel-Escalada et al . ( b ) Top 30 significantly enriched GO terms or gene sets for p65 footprints identified within the islet enhancer hubs. The footprint regions ( n =1684 within the 821 enhancer hubs) were assigned to the nearest transcription start site to perform GO term ontology and gene set enrichment using the ChIP-Enrich tool

Article Snippet: Membranes were incubated using standard techniques, diluents and buffers with anti-A20 (56305/D13H3), anti-IκBα (9242), anti-phospho-IκBα (2859/I4D4), anti-IKKγ (2585), anti-JNK (9252), anti-phospho-JNK (9255), anti-p65 (6956/L8F6) all sourced from Cell Signaling Technology, anti-mCherry (ab183628) (Abcam) or anti-β-actin (AC-15) (Sigma-Aldrich) antibodies, followed by labelling with the HRP-conjugated secondary antibody goat-anti-mouse IgG Fc (Pierce Antibodies) or donkey-anti-rabbit IgG (GE Life Sciences).

Techniques: Binding Assay

Journal: Diabetologia

Article Title: RELA governs a network of islet-specific metabolic genes necessary for beta cell function

doi: 10.1007/s00125-023-05931-6

Figure Lengend Snippet: SLC2A2 and CAPN9 locuses form part of the islet enhancer hubs linked with p65 regulation. ( a , b ) p65 ChIP-seq enrichment, epigenomic annotations and high-confidence pcHi-C interactions from islet samples at the two candidate loci: ( a ) SLC2A2 and ( b ) CAPN9 . p65 ChIP-seq data were obtained from ENCODE. Islet pcHi-C interactions and super enhancers were obtained from Miguel-Escalada et al . All browser views were generated using the WashU Epigenome Browser . Only the significant pcHi-C loops within the broadcast region are shown. ChIP, chromatin immunoprecipitation. ( c , d ) Real-time PCR analysis of ( c ) Slc2a2 and ( d ) Capn9 in islets isolated from p65fl/fl and βp65KO mice. Statistical analysis was performed using Student’s t tests. Data are means ± SEM. * p <0.05, *** p <0.001

Article Snippet: Membranes were incubated using standard techniques, diluents and buffers with anti-A20 (56305/D13H3), anti-IκBα (9242), anti-phospho-IκBα (2859/I4D4), anti-IKKγ (2585), anti-JNK (9252), anti-phospho-JNK (9255), anti-p65 (6956/L8F6) all sourced from Cell Signaling Technology, anti-mCherry (ab183628) (Abcam) or anti-β-actin (AC-15) (Sigma-Aldrich) antibodies, followed by labelling with the HRP-conjugated secondary antibody goat-anti-mouse IgG Fc (Pierce Antibodies) or donkey-anti-rabbit IgG (GE Life Sciences).

Techniques: ChIP-sequencing, Generated, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Isolation

Journal: Diabetologia

Article Title: RELA governs a network of islet-specific metabolic genes necessary for beta cell function

doi: 10.1007/s00125-023-05931-6

Figure Lengend Snippet: Annotation of p65 binding sites in proximal promoters and enhancers of glycolytic genes, with impacts on gene expression

Article Snippet: Membranes were incubated using standard techniques, diluents and buffers with anti-A20 (56305/D13H3), anti-IκBα (9242), anti-phospho-IκBα (2859/I4D4), anti-IKKγ (2585), anti-JNK (9252), anti-phospho-JNK (9255), anti-p65 (6956/L8F6) all sourced from Cell Signaling Technology, anti-mCherry (ab183628) (Abcam) or anti-β-actin (AC-15) (Sigma-Aldrich) antibodies, followed by labelling with the HRP-conjugated secondary antibody goat-anti-mouse IgG Fc (Pierce Antibodies) or donkey-anti-rabbit IgG (GE Life Sciences).

Techniques: Binding Assay