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: Molecular medicine reports

Article Title: Protective effect of diethylcarbamazine inhibits NF-κB activation in isoproterenol-induced acute myocardial infarction rat model through the PARP pathway.

doi: 10.3892/mmr.2017.6695

Figure Lengend Snippet: Figure 4. Protective effect of diethylcarbamazine inhibits inflammation response in isoproterenol‑induced AMI rats. Protective effect of diethyl carbamazine reduced (A) TNF‑α, (B) IL‑6 and (C) NF‑κB/p65 level in isoproterenol‑induced AMI rats. ##P<0.01 vs. control group and **P<0.01 vs. AMI model group. TNF, tumor necrosis factor; Control, control group; DEC, diethylcarbamazine‑alone group; AMI, acute myocardial infarction model group; AMI + DEC, AMI model + diethylcarbamazine treated group; IL, interleukin; NF‑κB, nuclear factor‑κB.

Article Snippet: The CK (A032, Nanjing Jiancheng Bioengineering Institute, Nanjing, China), LDH (E-EL-R0338c), tumor necrosis factor (TNF)-α (-EL-R0019c), interleukin (IL) -6 (E-EL-R0015c, Elabscience) and NF-κB/p65 (E-EL-R0674c) (all from Elabscience Biotechnology, Co., Ltd., Bethesda, MD, USA) activities were measured using commercial ELISA kits according to the manufacturer's protocols.

Techniques: Control

Journal: Materials Today Bio

Article Title: miR-19b-3p engineered adipose-derived stem cell exosomes attenuate acute liver failure via promoting tissue repair and microenvironment remodeling

doi: 10.1016/j.mtbio.2025.102697

Figure Lengend Snippet: miR-19-EXO dampened M1 macrophages polarization via inhibiting NF-κB canonical activation. (A) The procedure of THP1-based macrophage polarization assay. THP1 monocytes were treated with 100 ng/mL PMA for 48 h (for M0 induction), then treatment treated with 20 ng/mL LPS and 20 ng/mL IFN-γ for M1 subtype polarization, or 20 ng/mL IL4 and 20 ng/mL IL-13 or M2 subtype polarization, respectively. Representative morphology of M0, M1, M2 were recorded (scale bar = 100 μm). (B) and (D) RT-PCR analysis of M1 marker TNF-α , Il-6 and M2 marker Il-10 , CD163 in WT-EXO/miR-19-EXO treated M1 or M2 cells. (C) and (E) The macrophage polarization status in WT-EXO/miR-19-EXO treated M1/M2 cells was estimated by CD86 + and CD206 + cells ration using flow cytometry. (F) Thp1-M1 cells treated with miR-19-EXO or miR-19KD-EXO (100 μg/mL) for 24 h, then protein level of p47phox, NF-κB P65 and p-P65 were examined by immunoblotting. Thp1-M0 as control. (G) Thp1-M1 cell administrated with miR-19-EXO (100 μg/mL) for 24 h or NC were treated with CHX (100 μg/mL) for 0, 30, 60, 120, 240 min. Immunoblotting for iKBα expression levels in whole-cell extraction. (H) Thp1-M1 cell administrated with miR-19-EXO for 24 h or NC were treated with MG132 (10 mmol/L) for an extra 12h, then immunoblotting for iKBα protein levels in each group. (I) Thp1-M1 cell administrated with miR-19-EXO for 24 h or NC were immunoprecipitated with IgG control or iKBα antibody and then immunoblotted for Ubiquitin and iKBα. (J) Thp1-M1 cells treated with miR-19-EXO for 24 h were subjected to nuclear cytoplasmic separation assay, NF-κB P65 and IKKα level and translocation were examined by immunoblotting. LaminB1 was used as an internal control of nuclear protein, GAPDH as cytoplasmic protein control. (K) Schematic diagram for the mechanisms of IKK/NF-κB pathway activation suppressed by miR-19-EXO. (L)Thp1-M1 cells treated with miR-19-EXO or Thp1-M1 shp47phox cells or Thp1-M1 shp47phox cells treated with miR-19-EXO (100 μg/mL) for 24 h were detected the protein level of p47phox, NF-κB P65 and p-P65 by immunoblotting. Thp1-M1 as control (Ctrl). (M)Statistical differences were determined using unpaired Student's t -test between each 2 cohorts (ns = no significance, ∗p < 0.05, ∗∗p < 0.01).

Article Snippet: Next, the protein A/G agarose beads were pre-binded with 500 μL of anti-iKBα antibody (1:250, Proteintech, China) or rabbit IgG antibody (1:250, CST, USA) by rotating 1h at room temperature, then washed the antibody connected-beads for 3 times.

Techniques: Activation Assay, Reverse Transcription Polymerase Chain Reaction, Marker, Flow Cytometry, Western Blot, Control, Expressing, Extraction, Immunoprecipitation, Ubiquitin Proteomics, Translocation Assay