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: Oncogene

Article Title: Divergent C-terminal transactivation domains of Rel/NF-kappa B proteins are critical determinants of their oncogenic potential in lymphocytes.

doi: 10.1038/sj.onc.1207221

Figure Lengend Snippet: Figure 1 Mammalian c-Rel proteins transform primary spleen cells to induce colony formation in soft agar. (a) Transformation assay of primary lymphoid cells by v-Rel, chicken c-Rel or mammalian c-Rel, RelA, and IKKbSS-EE. Chicken spleen cells were electroporated with pJD214-derived retroviral vectors encoding individual Rel, RelA, or IKKbSS-EE proteins along with SW253 helper virus DNA. Colonies of transformed lymphoid cells were scored 2 weeks post-transfection. Results are shown for three independent experiments, each performed in duplicate. (b) Immunoblot analysis of Rel protein expression. Extracts from 293 T cells transiently transfected with pJD214-v-rel, -cc-rel, -mc-rel, -hc-rel, -hrelA, or an empty pJD214 vector control were resolved by SDS–PAGE, and analysed by immunoblotting with an antibody against the common N-terminal Rel homology domain (RHD) of Rel proteins (sc-6955; lanes 1–5) or an antibody against the N-terminus of hRelA (1207). An asterisk marks the position of endogenous RelA. (c) Immunoblot analysis of viral and cellular Rel/NF-kB protein expression in stably transformed spleen cell clones. Extracts from independent colonies of transformed spleen cells were resolved by SDS–10% PAGE and analysed by immunoblotting with an antibody that recognizes the common N-terminal RHD of Rel proteins (sc-6955)

Article Snippet: Commercially available antibodies were polyclonal anti-human c-Rel N-terminus (sc-272) or monoclonal anti-human c-Rel Nterminus antibody (sc-6955) (Santa Cruz Biotechnology, CA, USA), polyclonal anti-human RelA C-terminus (100–4165; Rockland Immunochemicals, Gilbertsville, PA, USA), and anti-actin (Sigma).

Techniques: Transformation Assay, Derivative Assay, Retroviral, Virus, Transfection, Western Blot, Expressing, Plasmid Preparation, Control, SDS Page, Stable Transfection, Clone Assay

Journal: Oncogene

Article Title: Divergent C-terminal transactivation domains of Rel/NF-kappa B proteins are critical determinants of their oncogenic potential in lymphocytes.

doi: 10.1038/sj.onc.1207221

Figure Lengend Snippet: Figure 2 Spleen cells expressing mammalian c-Rel proteins induce tumors in vivo. Individual colonies of primary spleen cells transformed by the mouse or human c-Rel proteins, or the RelA/ v-Rel hybrid were expanded in liquid culture and assayed for tumor formation by intraperitoneal transplantation of cells (2 106) into 5-day-old chickens. v-Rel-transformed cells were used as control. Survival represents the number of live chickens and is plotted relative to the number of days postinoculation. All animals that succumbed had developed visible tumor nodules on their spleen and liver at the time of autopsy

Article Snippet: Commercially available antibodies were polyclonal anti-human c-Rel N-terminus (sc-272) or monoclonal anti-human c-Rel Nterminus antibody (sc-6955) (Santa Cruz Biotechnology, CA, USA), polyclonal anti-human RelA C-terminus (100–4165; Rockland Immunochemicals, Gilbertsville, PA, USA), and anti-actin (Sigma).

Techniques: Expressing, In Vivo, Transformation Assay, Transplantation Assay, Control

Journal: Oncogene

Article Title: Divergent C-terminal transactivation domains of Rel/NF-kappa B proteins are critical determinants of their oncogenic potential in lymphocytes.

doi: 10.1038/sj.onc.1207221

Figure Lengend Snippet: Figure 3 DNA-binding and transcriptional activities of Rel and RelA hybrid proteins. (a) Schematic representation of wild-type and chimeric Rel and RelA proteins. In hybrid proteins, amino-acid numbers correspond to those of the parental wild type. RHD, Rel homology domain; NLS, nuclear localization signal; TAD, transactivation domain. The arrow indicates the junction at which hybrid proteins were generated. The NLS is shown as a black box; v-Rel envelope-derived sequences are shown as a gray box. (b) Western-blot analysis of hybrid protein expression. Extracts from 293T cells transiently transfected with pJD214-derived retroviral vectors encoding wild-type or chimeric Rel and RelA proteins or an empty pJD214 vector control were resolved by SDS–PAGE and analysed by immunoblotting with antibodies to v-Rel, c-Rel, or RelA as indicated. Arrows point to the hybrid proteins; asterisks mark the position of endogenous hRelA. (c) DNA-binding activity of hybrid Rel and RelA proteins. Nuclear extracts (3 mg) from 293T cells transiently transfected with pJD214 retroviral vectors encoding wild-type or hybrid proteins were assayed for binding to a double-stranded 32P- labeled IL-6-kB oligonucleotide probe. DNA/protein complexes were analysed on a native 5% polyacrylamide gel. Where indicated, antibodies to v-Rel-N (1967), v-Rel-C (1691), RelA-N (1207), or RelA-C (4165) were added to supershift DNA/protein complexes. Arrows point to DNA-bound complexes; brackets mark supershifted complexes. (d) Transcriptional activity of hybrid Rel and RelA proteins. Tera-2 cells (2 105) were transiently transfected with JD214-derived vectors expressing wild-type or hybrid proteins (0.8 mg), IL-6-kB-luciferase reporter (0.4 mg), and pRL-null internal luciferase control (0.0125 mg). Dual luciferase assays were performed at 48 h post-transfection. Results represent the average of three independent experiments performed in duplicate. The transcriptional activity of v-Rel and c-Rel proteins is highlighted in the magnified inset

Article Snippet: Commercially available antibodies were polyclonal anti-human c-Rel N-terminus (sc-272) or monoclonal anti-human c-Rel Nterminus antibody (sc-6955) (Santa Cruz Biotechnology, CA, USA), polyclonal anti-human RelA C-terminus (100–4165; Rockland Immunochemicals, Gilbertsville, PA, USA), and anti-actin (Sigma).

Techniques: Binding Assay, Generated, Derivative Assay, Western Blot, Expressing, Transfection, Retroviral, Plasmid Preparation, Control, SDS Page, Activity Assay, Labeling, Luciferase

Journal: Oncogene

Article Title: Divergent C-terminal transactivation domains of Rel/NF-kappa B proteins are critical determinants of their oncogenic potential in lymphocytes.

doi: 10.1038/sj.onc.1207221

Figure Lengend Snippet: Figure 4 Substitution with the transactivation domain of v-Rel confers potent oncogenic activity to RelA. (a) Transformation of primary lymphoid cells with chimeric Rel and RelA proteins. Primary spleen cells were transformed as described for Figure 1a. Results are shown for three independent experiments, one performed in duplicate and two in triplicate. The asterisk for the mc-Rel/hRelA hybrid indicates small abortive colonies that failed to grow in liquid culture. (b) Immunoblot analysis of the hybrid RelA/v-Rel protein expressed in stably transformed spleen cell clones. Extracts from independent colonies of transformed spleen cells were resolved by SDS–10% PAGE and analysed by immunoblotting with polyclonal antibodies specific for the N- terminus of RelA (1207) or the unique C-terminus of v-Rel (1691)

Article Snippet: Commercially available antibodies were polyclonal anti-human c-Rel N-terminus (sc-272) or monoclonal anti-human c-Rel Nterminus antibody (sc-6955) (Santa Cruz Biotechnology, CA, USA), polyclonal anti-human RelA C-terminus (100–4165; Rockland Immunochemicals, Gilbertsville, PA, USA), and anti-actin (Sigma).

Techniques: Activity Assay, Transformation Assay, Western Blot, Stable Transfection, Clone Assay

Journal: Oncogene

Article Title: Divergent C-terminal transactivation domains of Rel/NF-kappa B proteins are critical determinants of their oncogenic potential in lymphocytes.

doi: 10.1038/sj.onc.1207221

Figure Lengend Snippet: Figure 5 The transactivation domains of the human and mouse c-Rel proteins differ in oncogenic capacity. (a) Schematic representation of wild-type and chimeric Rel and RelA proteins. In the hybrids, amino-acid numbers correspond to those of the wild type. The arrow indicates the junction at which chimeric proteins were generated. The NLS is shown as a black box; v-Rel envelope- derived sequences are shown as a gray box. (b) Transformation of primary spleen cells with hybrid Rel and RelA proteins. Cells were transformed as described for Figure 1a. Results are shown for three independent experiments, of which one was performed in duplicate and another in triplicate. (c) Detection of hybrid Rel and RelA proteins in stably transformed spleen cell clones. Extracts from independent colonies of transformed cells were analysed by immunoblotting with antibodies specific for the common N-terminus of c- Rel proteins (sc-6955), the N-terminus of RelA (1207), or the C-terminus of mc-Rel (1266) as indicated. (d) Analysis of endogenous IkBa expression and association with mammalian c-Rel proteins. Extracts from spleen cell clones transformed by the mouse or human c-Rel proteins were loaded directly on a gel (40 mg; lanes 1 and 2) or immunoprecipitated (400 mg) with anti-chicken IkBa antibody (1275; lanes 3 and 4). After transfer, samples were immunoblotted with antibodies that recognize the N-terminal Rel-homology domain (sc-6955, top panel) or the endogenous chicken IkBa protein (1275; middle panel). Lanes containing crude extracts were reprobed with an anti-actin-antibody (bottom panel) as a control

Article Snippet: Commercially available antibodies were polyclonal anti-human c-Rel N-terminus (sc-272) or monoclonal anti-human c-Rel Nterminus antibody (sc-6955) (Santa Cruz Biotechnology, CA, USA), polyclonal anti-human RelA C-terminus (100–4165; Rockland Immunochemicals, Gilbertsville, PA, USA), and anti-actin (Sigma).

Techniques: Generated, Derivative Assay, Transformation Assay, Stable Transfection, Clone Assay, Western Blot, Expressing, Immunoprecipitation, Control

Journal: Oncogene

Article Title: Divergent C-terminal transactivation domains of Rel/NF-kappa B proteins are critical determinants of their oncogenic potential in lymphocytes.

doi: 10.1038/sj.onc.1207221

Figure Lengend Snippet: Figure 6 The v-Rel transactivation domain selectively confers an oncogenic phenotype upon the RHD of RelA but not all cellular Rel/NF-kB factors. (a) Schematic representation of wild-type and chimeric Rel and NF-kB proteins. In the hybrids, amino-acid numbers correspond to those of the wild type. The arrow indicates the junction at which chimeric proteins were generated. The NLS is shown as a black box; v-Rel envelope-derived sequences are shown as a gray box. (b) Transformation of primary spleen cells with hybrid Rel and NF-kB proteins. Cells were transformed as described for Figure 1a. Results are shown for three independent experiments. The asterisk for mRelB indicates a small abortive colony that failed to grow in liquid culture. ND, not determined

Article Snippet: Commercially available antibodies were polyclonal anti-human c-Rel N-terminus (sc-272) or monoclonal anti-human c-Rel Nterminus antibody (sc-6955) (Santa Cruz Biotechnology, CA, USA), polyclonal anti-human RelA C-terminus (100–4165; Rockland Immunochemicals, Gilbertsville, PA, USA), and anti-actin (Sigma).

Techniques: Generated, Derivative Assay, Transformation Assay

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: FEBS Open Bio

Article Title: Trastuzumab induces PUMA ‐dependent apoptosis and inhibits tumor growth in gastric cancer

doi: 10.1002/2211-5463.12522

Figure Lengend Snippet: p65 mediates trastuzumab‐induced PUMA induction. (A) NCI ‐N87 cells were treated with 10 μmol·L −1 trastuzumab at indicated time point. Indicated protein expression was analyzed by western blotting. (B) NCI ‐N87 cells were treated with 10 μmol·L −1 trastuzumab at indicated time point. p‐p65 (S536) and p65 expression was analyzed by western blotting. (C) NCI ‐N87 cells were transfected with either a control scrambled si RNA or a p65 si RNA for 24 h and then treated with 10 μmol·L −1 trastuzumab for 24 h. p65 and PUMA expression was analyzed by western blotting. (D) NCI ‐N87 cells were pretreated with 10 μmol·L −1 BAY 11‐7082 for 1 h and then with 10 μmol·L −1 trastuzumab for 24 h. Nuclear fractions were isolated from cells and analyzed for p65 expression by western blotting. Lamin A/C and β‐actin were used as controls for loading and fractionation. (E) NCI ‐N87 cells were pretreated with 10 μmol·L −1 BAY 11‐7082 for 1 h and then with 10 μmol·L −1 trastuzumab for 24 h. p‐p65 (S536) and PUMA expression was analyzed by western blotting. (F) Chromatin immunoprecipitation (Ch IP ) was performed using anti‐p65 antibody on NCI ‐N87 cells following trastuzumab treatment for 12 h. Ch IP with the control IgG was used as a control. PCR was carried out using primers surrounding the p65 binding sites in the PUMA promoter.

Article Snippet: SiRNA for scrambled control, for p65 (sc‐29410), as well as for GSK3 β (sc‐35527) were obtained from Santa Cruz Biotechnology.

Techniques: Expressing, Western Blot, Transfection, Control, Isolation, Fractionation, Chromatin Immunoprecipitation, Binding Assay

Journal: FEBS Open Bio

Article Title: Trastuzumab induces PUMA ‐dependent apoptosis and inhibits tumor growth in gastric cancer

doi: 10.1002/2211-5463.12522

Figure Lengend Snippet: The PUMA induction by trastuzumab is mediated through GSK 3β activation. (A) NCI ‐N87 cells were transfected with either a control scrambled si RNA or a GSK 3 β si RNA for 24 h and then treated with 10 μmol·L −1 trastuzumab for 6 h. Nuclear fractions were isolated from cells treated with trastuzumab and analyzed for p65 and GSK 3β expression by western blotting. (B) NCI ‐N87 cells were transfected with either a control scrambled si RNA or a GSK 3 β si RNA for 24 h and then treated with 10 μmol·L −1 trastuzumab for 24 h. GSK 3β and PUMA expression was analyzed by western blotting. (C) NCI ‐N87 cells were treated with 10 μmol·L −1 trastuzumab at indicated time point. Relative protein expression was analyzed by western blotting. (D) NCI ‐N87 cells were transfected with active AKT plasmid for 8 h and then treated with 10 μmol·L −1 trastuzumab for 24 h. PUMA , p‐ AKT , and total AKT expression was analyzed by western blotting.

Article Snippet: SiRNA for scrambled control, for p65 (sc‐29410), as well as for GSK3 β (sc‐35527) were obtained from Santa Cruz Biotechnology.

Techniques: Activation Assay, Transfection, Control, Isolation, Expressing, Western Blot, Plasmid Preparation

Journal: FEBS Open Bio

Article Title: Trastuzumab induces PUMA ‐dependent apoptosis and inhibits tumor growth in gastric cancer

doi: 10.1002/2211-5463.12522

Figure Lengend Snippet: The PUMA mediates the antitumor effects of trastuzumab in a xenograft model. (A) Nude mice were injected s.c. with 4 × 10 6 parental and PUMA ‐ KD NCI ‐N87 cells. After 1 week, mice were treated with 3 mg·kg −1 trastuzumab or buffer. Tumor volume at indicated time points after treatment was calculated and plotted ( n = 7 in each group). Results were expressed as means ± SD of 3 independent experiments. ** P < 0.01; *, P < 0.05 (Student's t ‐test). (B) Parental NCI ‐N87 xenograft tumors were treated with 3 mg·kg −1 trastuzumab or the control buffer as in (A). Phospho‐p65 (S536) and PUMA in representative tumors were analyzed by western blotting. (C) Paraffin‐embedded sections of tumor tissues from mice treated as in (A) were analyzed by TUNEL staining. Representative staining pictures (left) and TUNEL ‐positive cells (right) were counted and plotted. Scale bar, 50 μm. Results were expressed as means ± SD of 3 independent experiments. ** P < 0.01 (Student's t ‐test).

Article Snippet: SiRNA for scrambled control, for p65 (sc‐29410), as well as for GSK3 β (sc‐35527) were obtained from Santa Cruz Biotechnology.

Techniques: Injection, Control, Western Blot, TUNEL Assay, Staining

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: JCI Insight

Article Title: NF- κ B represses retinoic acid receptor–mediated GPRC5A transactivation in lung epithelial cells to promote neoplasia

doi: 10.1172/jci.insight.153976

Figure Lengend Snippet: ( A ) Small interfering RNA (siRNA) targeting p65 and scramble control siRNA were transfected to Calu-1 cells, treated with or without TNF-α. The protein levels of GPRC5A and p65 were determined by Western blotting. ( B ) Calu-1 cells were transfected with plasmid overexpressing IκBα-AA mutant (S32A, S36A) or vector control, then treated with or without TNF-α; GPRC5A and IκBα protein levels were determined with specific antibodies through Western blotting. ( C ) Schematic representation of WT and truncation mutant of RelA/p65. ( D ) Calu-1 transfectants harboring vector control or inducible expression of FL and truncated p65 were established; cells were treated with doxycycline (300 ng/mL) for 24 hours, and GPRC5A protein levels were analyzed by Western blotting. These blots were run in separate gels performed in parallel with equal loading (please see uncropped/unedited gels in the supplement). ( E and F ) Calu-1 cells with inducible expression of WT p65 and serine 276A mutant were treated with doxycycline (300 ng/mL) for 24 hours; GPRC5A protein and mRNA levels were determined by Western blotting ( E ) and qPCR ( F ), respectively. Data are presented as mean ± SD from 3 independent experiments with duplicates and analyzed by 2-tailed Student’s t test, *** P < 0.001.

Article Snippet: LPS was purchased from MilliporeSigma; TNF-α was purchased from R&D Systems; doxycycline was purchased from Selleckchem; antibodies (catalog numbers in parentheses) against H3K9ac (ab4441) and myc-tag (ab9232) for ChIP were purchased from Abcam; antibodies against Gprc5a (sc-98885), IκBα (sc-371), GAPDH (sc-365062), RARα (C-20), RARβ (C-19), and GFP (sc-8334) were purchased from Santa Cruz Biotechnology; NF-κB p65 NLS antibody (for IHC, NBP2-24541) was purchased from Novus Biologicals; antibodies against p-p65 (S536; 3033), H3K27Ac (8173), and β-actin conjugated with HRP (12262) were purchased from Cell Signaling Technology; p-p65 (S276) antibody (D155005) was from BBI Life Sciences Corporation; RNA polymerase II antibody (05-623B) was from MilliporeSigma; antibodies against myc-tag (catalog 562-5) and Flag-tag (catalog PM020) were purchased from MBL International Corporation; and customized GPRC5A antibody was generated by Abmart.

Techniques: Small Interfering RNA, Control, Transfection, Western Blot, Plasmid Preparation, Mutagenesis, Expressing

Journal: JCI Insight

Article Title: NF- κ B represses retinoic acid receptor–mediated GPRC5A transactivation in lung epithelial cells to promote neoplasia

doi: 10.1172/jci.insight.153976

Figure Lengend Snippet: ( A and B ) Calu-1 cells were treated with TNF-α and CHX separately or in combination; IκBα protein and mRNA levels were determined by Western blotting and quantified with ImageJ (NIH) ( A ) and qPCR ( B ), respectively. Data are presented as the mean ± SD. ( C and D ) Calu-1 cells were treated with TNF-α and CHX separately or in combination; GPRC5A mRNA and protein levels were determined by qPCR ( C ) and Western blotting and quantified with ImageJ ( D ), respectively. Data are presented as the mean ± SD. ( E ) Three NF-κB binding sites (designated as letters A–C) on GPRC5A promoter–luc plasmid were mutated individually or in combination. The repression effect of p65 was determined by luciferase assay. ( F ) The RA response element (RARE) at the GPRC5A promoter–luc plasmid was mutated, and the repression effect of p65 was determined by luciferase assay. Data are presented as mean ± SD from 3 independent experiments with duplicates and analyzed by 2-tailed Student’s t test, * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: LPS was purchased from MilliporeSigma; TNF-α was purchased from R&D Systems; doxycycline was purchased from Selleckchem; antibodies (catalog numbers in parentheses) against H3K9ac (ab4441) and myc-tag (ab9232) for ChIP were purchased from Abcam; antibodies against Gprc5a (sc-98885), IκBα (sc-371), GAPDH (sc-365062), RARα (C-20), RARβ (C-19), and GFP (sc-8334) were purchased from Santa Cruz Biotechnology; NF-κB p65 NLS antibody (for IHC, NBP2-24541) was purchased from Novus Biologicals; antibodies against p-p65 (S536; 3033), H3K27Ac (8173), and β-actin conjugated with HRP (12262) were purchased from Cell Signaling Technology; p-p65 (S276) antibody (D155005) was from BBI Life Sciences Corporation; RNA polymerase II antibody (05-623B) was from MilliporeSigma; antibodies against myc-tag (catalog 562-5) and Flag-tag (catalog PM020) were purchased from MBL International Corporation; and customized GPRC5A antibody was generated by Abmart.

Techniques: Western Blot, Binding Assay, Plasmid Preparation, Luciferase

Journal: JCI Insight

Article Title: NF- κ B represses retinoic acid receptor–mediated GPRC5A transactivation in lung epithelial cells to promote neoplasia

doi: 10.1172/jci.insight.153976

Figure Lengend Snippet: ( A ) RARβ in H157 cells was knocked down by siRNA followed by treatment with or without ATRA. The mRNA levels of RARβ and GPRC5A were determined by RT-PCR and quantified by ImageJ. ( B and C ) RARβ in H157 cells was knocked down by RARβ short hairpin (sh) RNA followed by treatment with or without ATRA. Protein and mRNA levels of RARβ and GPRC5A were determined by Western blotting ( B ) and RT-PCR and quantified by ImageJ ( C ), respectively. ( D and E ) Calu-1 cells were treated with TNF-α and ATRA separately or in combination, and the GPRC5A protein and mRNA levels were determined by Western blotting ( D ) and RT-PCR and quantified by ImageJ ( E ). ( F ) H157 cells was treated with TNF-α and ATRA separately or in combination. GPRC5A mRNA levels were determined by RT-PCR and quantified by ImageJ. ( G ) HEK293T cells were transfected with GPRC5A-luc and p65 plasmids and treated with or without ATRA. The p65 repression effect was determined by luciferase assay. Data are presented as mean ± SD from 3 independent experiments with duplicates and analyzed by 2-tailed Student’s t test, * P < 0.05; *** P < 0.001.

Article Snippet: LPS was purchased from MilliporeSigma; TNF-α was purchased from R&D Systems; doxycycline was purchased from Selleckchem; antibodies (catalog numbers in parentheses) against H3K9ac (ab4441) and myc-tag (ab9232) for ChIP were purchased from Abcam; antibodies against Gprc5a (sc-98885), IκBα (sc-371), GAPDH (sc-365062), RARα (C-20), RARβ (C-19), and GFP (sc-8334) were purchased from Santa Cruz Biotechnology; NF-κB p65 NLS antibody (for IHC, NBP2-24541) was purchased from Novus Biologicals; antibodies against p-p65 (S536; 3033), H3K27Ac (8173), and β-actin conjugated with HRP (12262) were purchased from Cell Signaling Technology; p-p65 (S276) antibody (D155005) was from BBI Life Sciences Corporation; RNA polymerase II antibody (05-623B) was from MilliporeSigma; antibodies against myc-tag (catalog 562-5) and Flag-tag (catalog PM020) were purchased from MBL International Corporation; and customized GPRC5A antibody was generated by Abmart.

Techniques: Reverse Transcription Polymerase Chain Reaction, Western Blot, Transfection, Luciferase

Journal: JCI Insight

Article Title: NF- κ B represses retinoic acid receptor–mediated GPRC5A transactivation in lung epithelial cells to promote neoplasia

doi: 10.1172/jci.insight.153976

Figure Lengend Snippet: ( A and B ) Calu-1 cells were treated with TNF-α (10 ng/mL) for various time points. The change of p65 and other proteins as indicated in the figure binding to the IκBα promoter ( A ) and GPRC5A promoter ( B ) was analyzed by chromatin immunoprecipitation (ChIP) assay using corresponding specific antibodies. Input as positive control and normal IgG (N IgG) as negative control. ( C and D ) The interaction of p65 with RARα ( C ) and RARβ2/β4 ( D ) was determined by immunoprecipitation (IP) assay. ( E ) RARα-Flag–expressing plasmid was cotransfected with GFP/GFP-p65/GFP-p65-S276A mutant to HEK293T cells. After 48 hours, cells were lysed by RIPA, and IP assay was performed to detect the interaction between RARα and WT RelA/p65 or serine 276A mutant. ( F ) Representative images of IF analysis of Calu-1 cells transfected with RARα-Flag plus GFP-p65 or GFP-p65-S276A as indicated. Scale bar: 10 μm.

Article Snippet: LPS was purchased from MilliporeSigma; TNF-α was purchased from R&D Systems; doxycycline was purchased from Selleckchem; antibodies (catalog numbers in parentheses) against H3K9ac (ab4441) and myc-tag (ab9232) for ChIP were purchased from Abcam; antibodies against Gprc5a (sc-98885), IκBα (sc-371), GAPDH (sc-365062), RARα (C-20), RARβ (C-19), and GFP (sc-8334) were purchased from Santa Cruz Biotechnology; NF-κB p65 NLS antibody (for IHC, NBP2-24541) was purchased from Novus Biologicals; antibodies against p-p65 (S536; 3033), H3K27Ac (8173), and β-actin conjugated with HRP (12262) were purchased from Cell Signaling Technology; p-p65 (S276) antibody (D155005) was from BBI Life Sciences Corporation; RNA polymerase II antibody (05-623B) was from MilliporeSigma; antibodies against myc-tag (catalog 562-5) and Flag-tag (catalog PM020) were purchased from MBL International Corporation; and customized GPRC5A antibody was generated by Abmart.

Techniques: Binding Assay, Chromatin Immunoprecipitation, Positive Control, Negative Control, Immunoprecipitation, Expressing, Plasmid Preparation, Mutagenesis, Transfection

Journal: JCI Insight

Article Title: NF- κ B represses retinoic acid receptor–mediated GPRC5A transactivation in lung epithelial cells to promote neoplasia

doi: 10.1172/jci.insight.153976

Figure Lengend Snippet: ( A ) GPRC5A protein expression level in multiple human NSCLC cell lines and normal human bronchial epithelial cell line (16HBE) was analyzed by Western blotting. ( B ) Calu-1 cells treated with or without TNF-α (10 ng/mL) for 12 hours. Binding of RNA polymerase II and the histone modification at the GPRC5A promoter were analyzed by ChIP using specific antibodies. ( C and D ) Calu-1 cells with inducible expression of WT and serine 276A mutant p65 were treated with doxycycline (300 ng/mL) for 12 hours; the change of RNA polymerase II binding ( C ) and histone modification (H3K9ac) at the GPRC5A promoter ( D ) were analyzed by ChIP. Input as positive control and normal IgG (N IgG) as negative control. ( E – H ) A549, H1975, and Calu-1 cells were treated with 5-Aza-dc (1 μM, 4 days) or SAHA (2.5 μM, 24 hours) individually or in combination; GPRC5A protein ( E – G ) and mRNA levels ( H ) were analyzed via Western blotting and qPCR. Data are presented as the mean ± SD. ( I ) Calu-1 cells were treated with or without SAHA (2.5 μM) for 12 hours; RNA polymerase II binding at the GPRC5A promoter was analyzed by ChIP. ( J ) Normal human bronchial epithelial cell line (16HBE) and multiple human NSCLC cell lines were treated with or without SAHA (2.5 μM, 24 hours); GPRC5A protein levels were analyzed by Western blotting. ( K ) Calu-1 cells were pretreated with DMSO (as vehicle control), 5-Aza-dc (1 μM, 3 days) or SAHA (2.5 μM, 3 hours) followed by TNF-α (10 ng/mL) treatment for an additional 24 hours. GPRC5A protein levels were analyzed by Western blotting. All data are presented as mean ± SD from 3 independent experiments with duplicates and analyzed by 2-tailed Student’s t test, ** P < 0.01; *** P < 0.001. H3K9ac, acetylated histone H3K9.

Article Snippet: LPS was purchased from MilliporeSigma; TNF-α was purchased from R&D Systems; doxycycline was purchased from Selleckchem; antibodies (catalog numbers in parentheses) against H3K9ac (ab4441) and myc-tag (ab9232) for ChIP were purchased from Abcam; antibodies against Gprc5a (sc-98885), IκBα (sc-371), GAPDH (sc-365062), RARα (C-20), RARβ (C-19), and GFP (sc-8334) were purchased from Santa Cruz Biotechnology; NF-κB p65 NLS antibody (for IHC, NBP2-24541) was purchased from Novus Biologicals; antibodies against p-p65 (S536; 3033), H3K27Ac (8173), and β-actin conjugated with HRP (12262) were purchased from Cell Signaling Technology; p-p65 (S276) antibody (D155005) was from BBI Life Sciences Corporation; RNA polymerase II antibody (05-623B) was from MilliporeSigma; antibodies against myc-tag (catalog 562-5) and Flag-tag (catalog PM020) were purchased from MBL International Corporation; and customized GPRC5A antibody was generated by Abmart.

Techniques: Expressing, Western Blot, Binding Assay, Modification, Mutagenesis, Positive Control, Negative Control, Control