Journal: Nature Communications

Article Title: NF-κB inhibition rescues cardiac function by remodeling calcium genes in a Duchenne muscular dystrophy model

doi: 10.1038/s41467-018-05910-1

Figure Lengend Snippet: NF-κB causes heart dysfunction in mdx mice. a EMSA performed on wild-type (wt) and mdx hearts (left panel). Supershift EMSA performed on mdx hearts using specific antibodies for p65 and p50, and IgG as a control (Right panel). Arrowheads indicate shifted bands. b Western blots performed on whole heart lysates and probed for phosphorylated p65-ser536 (phospho-p65), p65, p50, and α-tubulin (used as a loading control). c Representative images of H&E and phosho-p65 staining prepared from 1-year old heart sections. Boxed regions appear as magnified images in neighboring panels. Scale bar = 50 μm. d cardiac output, e stroke volume, f ejection fraction ( p = 0.686), g end diastolic diameter, and h end diastolic volume assessed by echocardiogram on 13–14-month-old mice ( n = 4 wt; 8 mdx IKKβf/f ; 5 mdx HRTΔIKKβ ). i End-diastolic pressure volume relationship (EDPVR) assessed by ventricular pressure-volume relationship analysis on 13–14-month old mice ( n = 7 wt; 18 mdx IKKβf/f ; 12 mdx HRTΔIKKβ ). j Developed force measured from isolated multicellular cardiac muscles of 7-month old mice in response to β-adrenergic stimulation with isoproterenol ( n = 7 wt; 7 mdx IKKβf/f ; 9 mdx HRTΔIKKβ ; 3 NBD treated ( mdx -NBD); p = 0.278). k Relaxation time (RT 90 ) after isoproterenol stimulation in multicellular cardiac muscles ( n = same as J). l, m Ventricular pressure-volume relationship measurements after dobutamine administration on 13–14-month-old mice. l Maximal heart rate (HR) ( n = 5 wt; 8 mdx IKKβf/f ; 7 mdx HRTΔIKKβ ) and ( m ) Tau (isovolumetric relaxation) ( n = 5 wt; 7 mdx IKKβf/f ; 4 mdx HRTΔIKKβ ; p = 0.027 but multiple comparisons test did not detect differences between groups). Data expressed as means ± SEM with bars and plungers and individual data points with dots. * p < 0.05 relative to wt and # p < 0.05 relative to mdx IKKβf/f by d – i , k – m 1-way ANOVA followed by Tukey multiple comparison test where appropriate, j 2-way repeated measures ANOVA. Main effects for genotype/treatment

Article Snippet: For supershifts, extracts were incubated with the following antibodies: 4 μl p50 (114 Santa Cruz) or IgG or 0.5 μl p65 (Rockland) prior to loading on the gel.

Techniques: Control, Western Blot, Staining, Isolation, Muscles, Comparison

Journal: Nature Communications

Article Title: NF-κB inhibition rescues cardiac function by remodeling calcium genes in a Duchenne muscular dystrophy model

doi: 10.1038/s41467-018-05910-1

Figure Lengend Snippet: Cardiomyocyte NF-κB ablation normalizes calcium handling and increases gene expression. a Statistically significant gene categories from microarray analysis identified using Gene Set Enrichment Analysis. Heatmaps represent genes identified in annotations. b Calcium transient amplitude measured from cardiomyocytes isolated from 7–8-month old mice ( n = 38 wt; 43 mdx IKKβf/f ; 35 mdx HRTΔIKKβ cardiomyocytes). c Depiction of individual microarray genes that were up- and down-regulated in mdx HRTΔIKKβ relative to mdx IKKβf/f hearts. Genes shown in red are ≥ 1.5-fold upregulated and those in blue are ≥ 1.5-fold downregulated. d – g qPCR analysis of Slc8a1 expression. RNA isolated from d 6–7-month-old hearts ( n = 5 wt; 5 mdx IKKβf/f ; 4 mdx HRTΔIKKβ ), e mouse embryonic fibroblasts (MEFs) that were wt ( p65 +/+ ) or null ( p65 −/− ) for p65 ( n = 5). f C2C12 myotubes expressing empty vector as control (CT) or IκBα super repressor (SR) ( n = 6 CT; 8 SR), and g MEFs untreated (CT) or treated with TNF ( n = 4). Data expressed as means ± SEM with bars and plungers and individual data points with dots. * p < 0.05 relative to respective control and # p < 0.05 relative to mdx IKKβf/f by b, d 1-way ANOVA followed by Tukey multiple comparison test and e – g 2-tailed Student’s t test

Article Snippet: For supershifts, extracts were incubated with the following antibodies: 4 μl p50 (114 Santa Cruz) or IgG or 0.5 μl p65 (Rockland) prior to loading on the gel.

Techniques: Gene Expression, Microarray, Isolation, Expressing, Plasmid Preparation, Control, Comparison

Journal: Nature Communications

Article Title: NF-κB inhibition rescues cardiac function by remodeling calcium genes in a Duchenne muscular dystrophy model

doi: 10.1038/s41467-018-05910-1

Figure Lengend Snippet: Cardiomyocyte NF-κB ablation causes global H3K27ac enrichment in mdx hearts. a Venn diagram pie chart depicting the H3K27ac ChIP-seq annotation within specified regions of the genome from mouse hearts. b Genome-wide distribution of H3K27ac binding loci relative to transcription start sites (TSS). c – d Genome-wide fragment density showing potential overlap of c . ChIP-seq histone marks across peaks from our ChIP-seq performed in mdx HRTΔIKKβ hearts and d our ChIP-seqs across peaks from p65 ChIP-seq. e Network showing the top 15 Gene Ontology clusters identified from differentially enriched genes in the mdx HRTΔIKKβ when compared to mdx IKKβf/f H3K27 regions. The most significantly enriched pathway for each cluster is labeled as the representative term for that group. f Gene expression analyzed by qPCR on total RNA isolated from hearts ( n = Rcan1 : 5 wt; 6 mdx IKKβf/f ; 6 mdx HRTΔIKKβ ; Cacna1h : 5 for all genotypes; Camk4 5 wt; 6 mdx IKKβf/f ; 5 mdx HRTΔIKKβ ). g – h ChIP performed with an H3K27ac antibody and qPCR analysis was used to detect enrichment on denoted genes. DNA extracted from g mouse hearts ( n = 3) or h control and TNF treated MEFs ( n = 4 Slc8a1 and Rcan1 and 3 Cacna1h and Camk4 ). Genes were expressed as a ratio. Dotted line represents level of enrichment equal to g mdx IKKβf/f hearts and h control MEFs. Bars represent ( g ) enrichment in hearts and h depletion in TNF treated MEFs. f Data expressed using box and whiskers plots. The central line in the boxes is the median value; the lower and upper boundaries of the boxes represent the lower and upper quartiles, respectively; the lower and upper whiskers represent the minimum and maximum values, respectively. Individual data points are plotted with dots. g – h Data expressed as means ± SEM with bars and plungers and individual data points with dots. f * p < 0.05 wt and # p < 0.05 mdx IKKβf/f , by 1-way ANOVA followed by Tukey multiple comparison test. g – h * p < 0.05 mdx IKKβf/f or untreated MEFs by 2-tailed Student’s t test

Article Snippet: For supershifts, extracts were incubated with the following antibodies: 4 μl p50 (114 Santa Cruz) or IgG or 0.5 μl p65 (Rockland) prior to loading on the gel.

Techniques: ChIP-sequencing, Genome Wide, Binding Assay, Labeling, Gene Expression, Isolation, Control, Comparison

Journal: Nature Communications

Article Title: NF-κB inhibition rescues cardiac function by remodeling calcium genes in a Duchenne muscular dystrophy model

doi: 10.1038/s41467-018-05910-1

Figure Lengend Snippet: CTCF, SIN3A, and HDAC1 mediate a less permissive chromatin conformation on calcium genes upon NF-κB activation. a Motif analysis performed on genes identified as having both p65 ChIP-seq peaks and H3K27ac mdx HRTΔIKKβ ChIP-seq regions. b Pie graph representing the percentage of genes with CTCF motifs up- and downregulated in the microarray (relative to mdx hearts with intact NF-κB). c ChIP-seq data derived from genome-wide fragment density analysis showing potential overlap of CTCF peaks with p65 peaks. d The same analysis as in c except p65 peaks were split between two groups either containing or lacking an NF-κB consensus motif. e – f ChIP performed with a CTCF antibody and qPCR analysis was used to detect enrichment on denoted genes. DNA extracted from e mdx IKKβf/f and mdx HRTΔIKKβ hearts ( n = 4 Slc8a1 ; 5 Rcan1 ; 3 cacna1h ; 2 Camk4 ) and ( f ) control and TNF treated MEFs ( n = 4 except n = 2 Camk4 ), g – h The same analyses were performed as in e, f . DNA extracted from mdx IKKβf/f and mdx HRTΔIKKβ hearts and ChIP performed with a ( g ) SIN3A antibody ( n = 3 Slc8a1 and Cacna1h ( p = 0.7); n = 4 Rcan1 and Camk4 ) ( h ) HDAC1 antibody ( n = 3 except n = 4 Slc8a1 ). e – h Enrichment on different genes were plotted as a ratio. Dotted line represents level of enrichment equal to e, g – h mdx IKKβf/f hearts and ( f ) control MEFs. Bars represent e, g – h depletion in mdx HRTΔIKKβ hearts and f enrichment in TNF treated MEFs. i Gene expression analyzed by qPCR on total RNA isolated from HL-1 cardiomyocytes ( n = 4). j Heatmaps showing ChIP-seq fragment densities of SIN3A and HDAC1 surrounding p65 peaks, showing potential overlap. Left panel includes genes upregulated and right panel includes genes downregulated in the microarray. e – h Data expressed as means ± SEM with bars and plungers and individual data points with dots. ( i ) Data expressed using box and whiskers plots. The central line in the boxes is the median value; the lower and upper boundaries of the boxes represent the lower and upper quartiles, respectively; the lower and upper whiskers represent the minimum and maximum values, respectively. Individual data points are plotted with dots. e – h * p < 0.05 mdx IKKβf/f or untreated by 2-tailed Student’s t test. i * p < 0.05 CT and # p < 0.05 TSA by 1-way ANOVA followed by Tukey multiple comparison test

Article Snippet: For supershifts, extracts were incubated with the following antibodies: 4 μl p50 (114 Santa Cruz) or IgG or 0.5 μl p65 (Rockland) prior to loading on the gel.

Techniques: Activation Assay, ChIP-sequencing, Microarray, Derivative Assay, Genome Wide, Control, Gene Expression, Isolation, Comparison

Journal: Cancer Cell International

Article Title: NF-κB and pSTAT3 synergistically drive G6PD overexpression and facilitate sensitivity to G6PD inhibition in ccRCC

doi: 10.1186/s12935-020-01576-2

Figure Lengend Snippet: p65 and pSTAT3 presented a synergistic effect on G6PD transcriptional regulation. a MatInspector software platform showed that the potential NF-κB- and STAT3-binding sites localized in the G6PD promoter region were adjacent to each other. Primers covering each indicated transcriptional factor–binding region were designed. b ChIP assay was performed with anti-p65 or p50/105 antibodies in ACHN, 786-O, and Caki-1 cells, and the eluate was amplified by real-time RT-PCR with primers covering the pSTAT3-binding site. c Similar experiments were repeated with anti-pSTAT3 or STAT3 antibody in ACHN786-O, and Caki-1 cells, and primers covering the NF-κB-binding site was used. d The interaction between pSTAT3 and p65 was determined by Co-IP in ACHN, 786-O, and Caki-1 cells. e–h The luciferase activity of G6PD-luc WT containing the NF-κB and pSTAT3 binding sites ( e , f ) and G6PD-luc Deletion without both the NF-κB- and pSTAT3-binding sequence ( g, h ) were analyzed in 293 T cells following treatment with the STAT3 or NF-κB signaling activator (IL-6, 2 ng/mL or TNFα, 50 ng/mL) ( e , g ), or inhibitor (STATTIC, 3 μM or BAY11-7082, 2.5 μM) ( f , h ) independently or jointly for 24 h. Data are expressed as mean ± SD from three independent experiments, each performed in triplicate. * P < 0.05, ** P < 0.01, *** P < 0.001, # P < 0.05, ## P < 0.01; ns, nonsignificant vs each control. ST, STATTIC; BAY, BAY11-7082

Article Snippet: Immunoprecipitation and Western blot analysis were carried out as described earlier using anti-phospho-STAT3 antibody (Ser 727) (ab30647, Abcam), anti-p65 antibody (ab32536, Abcam), or the control anti-immunoglobulin G antibody (X0936, DAKO A/S) [ ].

Techniques: Software, Binding Assay, Amplification, Quantitative RT-PCR, Co-Immunoprecipitation Assay, Luciferase, Activity Assay, Sequencing, Control

Journal: Cancer Cell International

Article Title: NF-κB and pSTAT3 synergistically drive G6PD overexpression and facilitate sensitivity to G6PD inhibition in ccRCC

doi: 10.1186/s12935-020-01576-2

Figure Lengend Snippet: NF-κB and STAT3 activated each other and facilitated ccRCC proliferation synergistically. a 786-O cells were treated with pSTAT3 stimulator IL-6 (4 ng/mL) or inhibitor STATTIC (6 μM) for 24 h. The changes in the expression of pSTAT3, STAT3, p50, p65, pIκBα, and IκBα at the protein level were detected using Western blot analysis. b 786-O or Caki-1 cells treated with TNFα (24 h) or BAY11-7082 (24 h) at indicated doses were subject to Western blot analysis to determine the protein expression changes of pSTAT3, STAT3, CyclinD1, and CDK4. 786-O or Caki-1 cells were infected with p65 RNAi lentivirus or negative control. The changes in the expression of STAT3, CyclinD1, and CDK4 at the mRNA level, and pSTAT3, STAT3, CyclinD1, and CDK4 expression at the protein level were determined by real-time RT-PCR c, d and Western blot ( e ) analysis, respectively. The relative proliferation rates of ACHN ( f ) or 786-O ( g ) cells following treatment with DMSO (control), STATTIC (pSTAT3 inhibitor, 6 μM), or BAY11-7082 (NF-κB inhibitor, 5 μM) were measured by MTS assay at indicated time course. ( H-I ) The control or G6PD-overexpressing ACHN cells were treated with STATTIC (6 μM) ( h ), or BAY11-7082 (5 μM) ( i ) for 0, 12, 24, and 36 h, and the relative proliferation rate was determined by MTS assay. j ACHN and 786-O cells were treated with STATTIC (6 μM) or BAY11-7082 (5 μM) independently or jointly for 36 h, and the relative proliferation rate was measured by MTS assay. k The control or G6PD-overexpressing ACHN cells were treated with DMSO or combination of STATTIC (6 μM) and BAY11-7802 (5 μM) for 36 h, and the relative proliferation rate was determined by MTS assay. β-Actin or GAPDH was used as a loading control. Data are expressed as mean ± SD from three independent experiments, each performed in triplicate. * P < 0.05, ** P < 0.01, *** P < 0.001, # P < 0.05, ## P < 0.01, and ### P < 0.001 vs each control. NC, negative control; ST, STATTIC; BAY, BAY11-7082; G6PD OE, G6PD overexpression

Article Snippet: Immunoprecipitation and Western blot analysis were carried out as described earlier using anti-phospho-STAT3 antibody (Ser 727) (ab30647, Abcam), anti-p65 antibody (ab32536, Abcam), or the control anti-immunoglobulin G antibody (X0936, DAKO A/S) [ ].

Techniques: Expressing, Western Blot, Infection, Negative Control, Quantitative RT-PCR, Control, MTS Assay, Over Expression

Journal: The Journal of allergy and clinical immunology

Article Title: ANKRD1 regulates innate immune responses against herpes simplex virus 1: A potential role in eczema herpeticum

doi: 10.1016/j.jaci.2018.01.001

Figure Lengend Snippet: The indicated expression plasmids were transfected into 293 FT cells and incubated overnight. The cells were then harvested and co-IP experiments were performed. Flag/DDK1-tagged protein expression and HA-tagged ANKRD1 protein expression are shown in IP samples and lysates. Data presented as one of three independent experiments.

Article Snippet: Myc-DDK1-tagged RELA (RC220780), Myc-DDK1-tagged NFKB1 (RC208384), Myc-DDK1-tagged IRF7 (RC217934), Myc-DDK1-tagged ANKRD1 (RC205609) and Myc-DDK1-tagged STING were purchased from OriGene (Rockville, MD).

Techniques: Expressing, Transfection, Incubation, Co-Immunoprecipitation Assay

Journal: Molecular medicine reports

Article Title: Helicobacter pylori infection enhances heparanase leading to cell proliferation via mitogen‑activated protein kinase signalling in human gastric cancer cells.

doi: 10.3892/mmr.2018.9558

Figure Lengend Snippet: Figure 2. H. pylori infection mediates the HPA protein expression increase in MKN‑45 cells via the MAPK signalling pathway. (A) Western blotting was used to detect the expression levels of p‑p38 MAPK, p38 MAPK, p‑p65 NF‑κB and p65 NF‑κB following different co‑culture durations. (B) p‑p38 MAPK protein expression. (C) p‑p65 NF‑κB protein expression levels were quantitatively analysed using densitometry. (D) Western blots and (E) quantitatively analysed expression of NF‑κB in MKN‑45 cells pre‑treated with SB203580 prior to co‑culture with H. pylori. *P<0.05 and **P<0.01, as indicated. MAPK, mitogen‑activated protein kinase; NF‑κB, nuclear factor‑κB; p‑, phosphorylated; H. pylori or Hp, Helicobacter pylori.

Article Snippet: Following the blocking step, the membranes were incubated with the following primary antibodies: anti-HPA1 (1:1,000; cat. no. ab128931), anti-phosphorylated (p)-p38 MAPK (1:1,000; cat. no. ab195049), anti-p38 MAPK (1:1,000; cat. no. ab170099), anti-p-p65 NF-κB (1:1,000; cat. no. ab76302), anti-p65 NF-κB (1:1,000; no. ab32536; all Abcam, Cambridge, UK) and anti-β-actin (1:2,000; cat. no. TA-09; OriGene Technologies, Inc., Beijing, China).

Techniques: Infection, Expressing, Western Blot