Journal: Immunity & Ageing : I & A

Article Title: Cell autonomous expression of inflammatory genes in biologically aged fibroblasts associated with elevated NF-kappaB activity

doi: 10.1186/1742-4933-5-5

Figure Lengend Snippet: NF-κB activity. Shown are the results of a DNA binding assay of the NF-κB p65 transcription factor, using two biological replicates and four readouts for each sample. NF-κB activity, a key mediator of inflammation, is elevated in the nuclear fractions of fibroblasts from the group of older donors compared to young donors (p < 0.005). The DNA binding activity corresponds to a "low-grade" inflammatory state compared to the TNFα treated positive control.

Article Snippet: 10 μg of the nuclear cell fractions were used from each cell-line and their NF-κB p65 DNA binding activity was determined using the EZ-Detect NF-κB p65 Transcription Factor Kit (Pierce Biotechnology, Product No. 89859, Rockford, IL) according to the manufacturer's protocol.

Techniques: Activity Assay, DNA Binding Assay, Binding Assay, Positive Control

Journal: Immunity & Ageing : I & A

Article Title: Cell autonomous expression of inflammatory genes in biologically aged fibroblasts associated with elevated NF-kappaB activity

doi: 10.1186/1742-4933-5-5

Figure Lengend Snippet: List of inflammatory genes. Given are accession numbers of genes, fold changes and p-values between the group of older and young donors, a Pearson correlation coefficient (R) expressing similarity to the NF-κB profile as seen in Figure 1, and a description of genes.

Article Snippet: 10 μg of the nuclear cell fractions were used from each cell-line and their NF-κB p65 DNA binding activity was determined using the EZ-Detect NF-κB p65 Transcription Factor Kit (Pierce Biotechnology, Product No. 89859, Rockford, IL) according to the manufacturer's protocol.

Techniques: Expressing, Variant Assay, Binding Assay

Journal: PLoS ONE

Article Title: The NF-κB Subunit RelA/p65 Is Dispensable for Successful Liver Regeneration after Partial Hepatectomy in Mice

doi: 10.1371/journal.pone.0046469

Figure Lengend Snippet: ( A ) Rela F/F and Rela F/F AlbCre animals were subjected to 2/3 PH and ALT serum levels were determined at the indicated time points. Data are expressed as mean ± SEM (n = 3−6 animals per time point and group). ( B ) No significant apoptosis was detected at any time point after PH in control or mutant mice as assessed by TUNEL-staining. Representative TUNEL stainings from control and mutant livers harvested 24 h post PH are shown in the upper row. Liver sections from TNF-injected (i.v. 10 ng/g BW) control and Rela F/F AlbCre animals which are highly sensitive to TNF-induced apoptosis served as control (lower panel, magnification ×200).

Article Snippet: For immunohistochemical analysis tissues were processed as described previously using anti-BrdU (Serotec) and goat anti-p65 (Santa Cruz).

Techniques: Control, Mutagenesis, TUNEL Assay, Staining, Injection

Journal: PLoS ONE

Article Title: The NF-κB Subunit RelA/p65 Is Dispensable for Successful Liver Regeneration after Partial Hepatectomy in Mice

doi: 10.1371/journal.pone.0046469

Figure Lengend Snippet: ( A–E ) 2/3 PH was performed in Rela F/F AlbCre and Rela F/F control animals. ( A ) DNA synthesis was assessed by hepatocyte BrdU labelling at the indicated time points and quantified as described in Methods (representative×200 anti-BrdU stained liver sections are shown in the left panel, n = 4−8 animals per time point and group were counted for quantification, right panel). ( B ) Accelerated cell cycle progression in Rela F/F AlbCre mice as determined by mRNA levels of Cyclin D1 24 h post PH (upper panel) and WB analysis of cell-cycle associated proteins (PCNA, Cyclin A, Cyclin D1). Lysates were controlled for effective deletion of WT-RelA/p65 and β-actin served as loading control. Two representative lysates from each time point post PH are shown. ( C ) Loss of hepatocyte RelA/p65 leads to enhanced phosphorylation of STAT3 and JNK during the first hours after PH as assessed by immunoblot analysis (upper panels). ( D ) Liver IL-6 mRNA induction as determined by RT-PCR (upper graph) and IL-6 serum levels (ELISA, lower graph) were not different between groups. ( E ) Liver mass regeneration (%) determined as described in Methods did not differ between the groups. Data from BrdU-labelling, cytokine analysis, RT-PCR, and analysis of liver mass regeneration are presented as the average ± SEM for 3–6 animals per time point per group. *, p≤0.05 for mutant vs. control mice.

Article Snippet: For immunohistochemical analysis tissues were processed as described previously using anti-BrdU (Serotec) and goat anti-p65 (Santa Cruz).

Techniques: Control, DNA Synthesis, Staining, Phospho-proteomics, Western Blot, Reverse Transcription Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Mutagenesis

Journal: PLoS ONE

Article Title: The NF-κB Subunit RelA/p65 Is Dispensable for Successful Liver Regeneration after Partial Hepatectomy in Mice

doi: 10.1371/journal.pone.0046469

Figure Lengend Snippet: ( A ) For extended (80%) PH the lower right lobe (LRL) and one omental lobe (OL) were additionally resected to standard 2/3 PH in control and Rela F/F AlbCre animals. ( B ) Survival of control and mutant animals did not differ between groups and was 4/6 in Rela F/F and 4/7 in Rela F/F AlbCre animals. ALT levels determined at 48 hours ( C ) and liver mass regeneration at 7 days post 80% PH ( D ).

Article Snippet: For immunohistochemical analysis tissues were processed as described previously using anti-BrdU (Serotec) and goat anti-p65 (Santa Cruz).

Techniques: Control, Mutagenesis

Journal: PLoS ONE

Article Title: The NF-κB Subunit RelA/p65 Is Dispensable for Successful Liver Regeneration after Partial Hepatectomy in Mice

doi: 10.1371/journal.pone.0046469

Figure Lengend Snippet: ( A ) WT RelA/p65 is deleted in Kupffer cells isolated from Rela F/F MxCre animals 7 days after one single i.p. injection of poly-IC (10 µg/g BW) as assessed be immunoblot analysis with anti-p65. β-actin served as loading control. ( B ) 45 min after i.p. TNF (25 ng/g BW) nuclear translocation of RelA/p65 is seen in hepatocytes (black arrowheads) and non-parenchymal cells (white arrowheads) in Rela F/F controls, confined to non-parenchymal cells in Rela F/F AlbCre animals and virtually absent in all liver cells in Rela F/F MxCre animals (magnification×200). (C) LPS (2 mg/kg) -induced TNF-response in mouse serum (left panel) and LPS-induced induction of the NF-κB target gene Tfnaip3 encoding A20 in the liver (right panel) is abolished in Rela F/F MxCre animals as assessed by ELISA and RT-PCR respectively. ( D ) A20 mRNA and IκBα mRNA expression as assessed by RT-PCR analysis at the indicated times post PH is inhibited in Rela F/F AlbCre animals but abolished in Rela F/F MxCre animals. Data from cytokine analysis and RT-PCR are presented as the average ± SEM for 3–6 animals per time point per group.

Article Snippet: For immunohistochemical analysis tissues were processed as described previously using anti-BrdU (Serotec) and goat anti-p65 (Santa Cruz).

Techniques: Isolation, Injection, Western Blot, Control, Translocation Assay, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: PLoS ONE

Article Title: The NF-κB Subunit RelA/p65 Is Dispensable for Successful Liver Regeneration after Partial Hepatectomy in Mice

doi: 10.1371/journal.pone.0046469

Figure Lengend Snippet: ( A–C ) 2/3 PH was performed on Rela F/F and Rela F/F MxCre animals and analyzed as indicated. ( A ) Levels of phospho-STAT3 in control and Rela F/F MxCre animals were not different while phosphorylation of JNK was barely detected in either group as assessed be immunoblot analysis. ( B ) Induction of liver IL-6 mRNA was inhibited in Rela F/F MxCre animals as determined by RT-PCR (upper image), however, IL-6 serum levels were significantly elevated in livers of Rela F/F MxCre mice at 4 h post PH (lower image). ( C ) Immunoblot analysis of cell cycle associated proteins was performed as described in Fig. 2. Data from cytokine analysis and RT-PCR are presented as the average ± SEM for 3–6 animals per time point per group. *, p≤0.05 for mutant vs. control mice.

Article Snippet: For immunohistochemical analysis tissues were processed as described previously using anti-BrdU (Serotec) and goat anti-p65 (Santa Cruz).

Techniques: Control, Phospho-proteomics, Western Blot, Reverse Transcription Polymerase Chain Reaction, Mutagenesis

Journal: PLoS ONE

Article Title: The NF-κB Subunit RelA/p65 Is Dispensable for Successful Liver Regeneration after Partial Hepatectomy in Mice

doi: 10.1371/journal.pone.0046469

Figure Lengend Snippet: ( A ) DNA-synthesis as determined by BrdU-uptake and ( B ) liver mass regeneration was not different in control and mutant animals. ( C ) Representative TUNEL staining at 24 h and ALT levels did not reveal significant apoptosis or altered liver damage in Rela F/F and Rela F/F MxCre animals. Data are presented as the average ± SEM for 3–6 animals per time point per group.

Article Snippet: For immunohistochemical analysis tissues were processed as described previously using anti-BrdU (Serotec) and goat anti-p65 (Santa Cruz).

Techniques: DNA Synthesis, Control, Mutagenesis, TUNEL Assay, Staining

Journal: iScience

Article Title: Hyperandrogenism triggers mtDNA release to participate in ovarian inflammation via mPTP/cGAS/STING in PCOS

doi: 10.1016/j.isci.2025.112391

Figure Lengend Snippet: Activation of inflammation in FF and GCs cells in patients with PCOS (A) Functional annotation for GCs-related genes using GO enrichment analysis ( n = 3). (B) Enriched pathways in KEGG analysis of GCs ( n = 3). (C) Levels of IL1B in FF ( n = 20). (D) Levels of IL17 in FF ( n = 20). (E) Levels of IL18 in FF ( n = 20). (F) mRNA levels of p65 in GCs in patients with PCOS and controls ( n = 10). (G) mRNA levels of IL1B in GCs in patients with PCOS and controls ( n = 10). (H) mRNA levels of IL18 in GCs in patients with PCOS and controls ( n = 10). (I) Quantification of protein expression levels of p-p65/p65 in GCs of patients with PCOS compared with controls ( n = 4). (J) Representative WB images of p-p65, p65, IL1B, and IL18 in GCs in patients with PCOS and controls. (K) Quantification of protein expression levels of IL1B in GCs of patients with PCOS compared with controls ( n = 4). (L) Quantification of protein expression levels of IL18 in GCs of patients with PCOS compared with controls ( n = 4). Data are represented as mean ± SEM.∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb , Cell Signaling Technology , Cat#3033s; RRID: AB_331284.

Techniques: Activation Assay, Functional Assay, Expressing

Journal: iScience

Article Title: Hyperandrogenism triggers mtDNA release to participate in ovarian inflammation via mPTP/cGAS/STING in PCOS

doi: 10.1016/j.isci.2025.112391

Figure Lengend Snippet: mtDNA release-induced activation of inflammation and cGAS-STING pathway in T-induced KGN cells in vitro (A) mRNA levels of cGAS, STING, p65, IL1B, and IL18 in KGN cells exposed to T compared with controls ( n = 3). (B) Representative western blotting images of p-p65, p65, IL1B, and IL18 in GCs of KGN cells exposed to T compared with controls. (C) Quantification of protein expression levels of p-p65/p65, IL1B, and IL18 in KGN cells exposed to T compared with controls ( n = 3). (D) Representative western blotting images of cGAS and STING in GCs in KGN cells exposed to T compared with controls. (E) Quantification of protein expression levels of cGAS and STNG in KGN cells exposed to T compared with controls ( n = 3). (F) RT-qPCR quantitative analysis of cytosolic mtDNA (D Loop) normalized to nuclear gene (B2M) of total lysates from the KGN cells exposed to T compared with controls ( n = 3). (G) RT-qPCR quantitative analysis of cytosolic mtDNA (ND1) normalized to nuclear gene (B2M) of total lysates from the KGN cells exposed to T compared with controls( n = 3). (H) Representative fluorescence images of cGAS in KGN cells exposed to T compared with controls (scale bar, 50 μm). (I) Quantitative analysis of fluorescence intensity for cGAS in KGN cells exposed to T compared with controls ( n = 3). (J) Representative western blotting images of cGAS, STING, p-p65, p65, IL1B, and IL18 after mtDNA transfection of KGN cells ( n = 3, mtDNA 1 μg/mL for 24 h). (K) Quantification of protein expression levels of p-p65/p65 after mtDNA transfection of KGN cells ( n = 3). (L) Quantification of protein expression levels of cGAS, STING, IL1B, and IL18 after mtDNA transfection of KGN cells ( n = 3). T, testosterone; Data are represented as mean ± SEM.∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb , Cell Signaling Technology , Cat#3033s; RRID: AB_331284.

Techniques: Activation Assay, In Vitro, Western Blot, Expressing, Quantitative RT-PCR, Fluorescence, Transfection

Journal: iScience

Article Title: Hyperandrogenism triggers mtDNA release to participate in ovarian inflammation via mPTP/cGAS/STING in PCOS

doi: 10.1016/j.isci.2025.112391

Figure Lengend Snippet: Remission of inflammation and follicle dysfunction following STING inhibition (A) mRNA levels of p65, IL1B, and IL18 in KGN cells exposed to T after STING knockdown by siRNA ( n = 3). (B) Representative western blotting images of p-p65, p65, IL1B, and IL18 in KGN cells exposed to T after STING knockdown by siRNA. (C) Quantification of protein expression levels of p-p65/p65, IL1B, and IL18 in KGN cells exposed to T after STING knockdown by siRNA ( n = 3). (D) Representative fluorescence images of p-p65 in KGN cells exposed to T after STING knockdown by siRNA (scale bar, 50 μm). (E) Quantitative analysis of fluorescence intensity for p-p65 in KGN cells exposed to T after STING knockdown by siRNA ( n = 3). (F) mRNA levels of NR3C4 in KGN cells exposed to T after STING knockdown by siRNA ( n = 3). (G) mRNA levels of FSHR in KGN cells exposed to T after STING knockdown by siRNA ( n = 3). (H) mRNA levels of HSD3B2 in KGN cells exposed to T after STING knockdown by siRNA ( n = 3). (I) mRNA levels of CYP11A1 in KGN cells exposed to T after STING knockdown by siRNA ( n = 3). T: testosterone; Data are represented as mean ± SEM.∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb , Cell Signaling Technology , Cat#3033s; RRID: AB_331284.

Techniques: Inhibition, Knockdown, Western Blot, Expressing, Fluorescence

Journal: iScience

Article Title: Hyperandrogenism triggers mtDNA release to participate in ovarian inflammation via mPTP/cGAS/STING in PCOS

doi: 10.1016/j.isci.2025.112391

Figure Lengend Snippet: Remission of inflammation and follicle dysfunction following inhibition of opening levels of mPTP (A) Representative fluorescence images of mPTP opening levels in KGN cells exposed to T and CsA (scale bar, 100 μm). (B) Quantitative analysis of fluorescence intensity for mPTP opening levels in KGN cells exposed to T and CsA ( n = 3). (C) RT-qPCR quantitative analysis of cytosolic mtDNA (D Loop) normalized to nuclear gene (B2M) of total lysates from the KGN cells exposed to T and CsA ( n = 3). (D) RT-qPCR analysis of cytosolic mtDNA (ND1) normalized to nuclear gene (B2M) of total lysates from the KGN cells exposed to T and CsA ( n = 3). (E) Representative western blotting images of p-p65, p65, and IL1B in KGN cells exposed to T and CsA. (F) Representative western blotting images of cGAS, STING, and IL18 in KGN cells exposed to T and CsA. (G) Quantification of protein expression levels of p-p65/p65 in KGN cells exposed to T and CsA ( n = 3). (H) Quantification of protein expression levels of cGAS, STING, IL1B and IL18 in KGN cells exposed to T and CsA ( n = 3). (I) mRNA levels of NR3C4 in KGN cells exposed to T and CsA ( n = 3). (J) mRNA levels of FSHR in KGN cells exposed to T and CsA ( n = 3). (K) mRNA levels of HSD3B2 in KGN cells exposed to T and CsA ( n = 3). (L) mRNA levels of CYP11A1 in KGN cells exposed to T and CsA ( n = 3). T: Testosterone, CsA: Cyclosporin A; Data are represented as mean ± SEM.∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb , Cell Signaling Technology , Cat#3033s; RRID: AB_331284.

Techniques: Inhibition, Fluorescence, Quantitative RT-PCR, Western Blot, Expressing

Journal: iScience

Article Title: Hyperandrogenism triggers mtDNA release to participate in ovarian inflammation via mPTP/cGAS/STING in PCOS

doi: 10.1016/j.isci.2025.112391

Figure Lengend Snippet: Alleviation of ovarian inflammation and follicle dysfunction after inhibition of STING in DHEA-induced mouse model (A–C) Representative H&E images of mouse ovary in the mouse injected DHEA and C176 (scale bar, 100 μm, DHEA 60 mg/kg per mouse daily, C176 750 nmol per mouse daily in 200 μL corn oil). (D–H) Quantification of follicles at all stages and corpus luteum in the mouse injected DHEA and C176 ( n = 6). (I) Quantification of body weight gain in the mouse injected DHEA and C176 ( n = 6). (J) mRNA levels of cGAS, STING, p65, IL1B, and IL18 in the ovarian tissue of mouse injected DHEA and C176 ( n = 3). (K) Quantification of the protein expression levels of p-p65/p65, IL1B, and IL18 in the ovarian tissue of mouse injected DHEA and C176 ( n = 3). (L) Representative western blotting images of p65, p-p65, IL1B, and IL18 in the ovarian tissue of mouse injected DHEA and C176 ( n = 3). (M) mRNA levels of NR3C4 in the ovarian tissue of mouse injected DHEA and C176 ( n = 3). (N) mRNA levels of FSHR in the ovarian tissue of mouse injected DHEA and C176 ( n = 3). (O) mRNA levels of HSD3B2 in the ovarian tissue of mouse injected DHEA and C176 ( n = 3). (P) mRNA levels of CYP11A1 in the ovarian tissue of mouse injected DHEA and C176 ( n = 3). Data are represented as mean ± SEM.∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001.

Article Snippet: Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb , Cell Signaling Technology , Cat#3033s; RRID: AB_331284.

Techniques: Inhibition, Injection, Expressing, Western Blot

Journal: iScience

Article Title: Hyperandrogenism triggers mtDNA release to participate in ovarian inflammation via mPTP/cGAS/STING in PCOS

doi: 10.1016/j.isci.2025.112391

Figure Lengend Snippet:

Article Snippet: Phospho-NF-κB p65 (Ser536) (93H1) Rabbit mAb , Cell Signaling Technology , Cat#3033s; RRID: AB_331284.

Techniques: Recombinant, Modification, Reverse Transcription, Immunostaining, Blocking Assay, DNA Extraction, Permeability, Sequencing, Software

Journal: Cancer Research

Article Title: Mutant p53 Enhances Nuclear Factor κB Activation by Tumor Necrosis Factor α in Cancer Cells

doi: 10.1158/0008-5472.can-06-2425

Figure Lengend Snippet: Figure 2. Nuclear p65 accumulation upon TNFa treatment is enhanced in the presence of mutant p53. A, SKBR3 cells were seeded on glass coverslips and transfected with synthetic p53 siRNA. Twenty-four hours later, cells were treated with 50 ng/mL TNFa for 30 min (TNF) or left untreated (NT). Slides were then fixed in 4% paraformaldehyde in PBS for 10 min, rinsed 3 with PBS, blocked for 30 min with PBS containing 0.1% Triton X-100, 5% goat serum, and 0.2% bovine serum albumin, and incubated overnight with either anti-p65 C-20 (Santa Cruz Biotechnology, Santa Cruz, CA) or a mixture of the p53-specific monoclonal antibodies PAb1801 and DO-1. Slides were washed 3 with PBS, incubated with secondary antibodies conjugated to Alexa 466 (Molecular Probes, Eugene, OR) or Cy3 (Chemicon, Temecula, CA) for 1 h, and visualized under a fluorescence microscope. B, SKBR3 cells were treated as in (A). Multiple fields were observed by fluorescence microscopy, and relative p53 and nuclear p65 levels were scored in parallel for each individual cell. Cells were segregated into two categories, based on residual p53 levels after knockdown. The distribution of relative nuclear p65 levels among cells of each category is shown. The data are based on counting a total of 324 cells, in four groups. Columns, means; bars, SD. C, cells from the same experiment as in (A) were fixed and stained after 3 h of treatment with TNFa. D, SKBR3 cells were treated as in (C). Multiple fields were observed by fluorescence microscopy, and relative p53 and nuclear p65 levels were scored in parallel for each individual cell. Cells were segregated in two categories, based on residual p53 levels after knockdown. The distribution of relative nuclear p65 levels among cells of each category is shown. The data are based on counting a total of 242 cells in four groups. Columns, means; bars, SD.

Article Snippet: Staining was essentially as in (A ), except that a polyclonal anti-phospho-p65 NFnB (Ser536) antibody (Cell Signaling Technology, Danvers, MA) was employed to monitor NFnB activation.

Techniques: Mutagenesis, Transfection, Incubation, Bioprocessing, Fluorescence, Microscopy, Knockdown, Staining

Journal: Cancer Research

Article Title: Mutant p53 Enhances Nuclear Factor κB Activation by Tumor Necrosis Factor α in Cancer Cells

doi: 10.1158/0008-5472.can-06-2425

Figure Lengend Snippet: Figure 4. Mutant p53 overexpression coincides with NFnB activation in human malignant and premalignant head and neck lesions. A, representative immunohistochemical results of NFnB p65 and p53 staining, on serial sections of paraffin-embedded specimens derived from human head and neck premalignant and malignant lesions. Formalin-fixed paraffin-embedded material, surgically resected from 15 patients with head and neck squamous cell carcinomas, some also containing adjacent hyperplastic and dysplastic lesions of the same patient on a single slide, was from a previously published series (20); the p53 status of these cases has been previously described (20). This group was supplemented with 10 new tumor specimens. The presence of mutant p53 was concluded on the basis of positive immunohistochemical staining for p53. In 12 of those cases, the presence of p53 mutations was confirmed by single-strand conformational polymorphism analysis (SCCP) and in three of those cases also by direct sequencing. Five of the immunohistochemistry-negative tumors were also subjected to SCCP analysis, and all five generated a pattern consistent with the presence of wt p53 only. Anti-p53 (DO-7; DAKO, Carpinteria, CA) and anti-NFnB p65 (F-6; Santa Cruz Biotechnology) monoclonal antibodies were employed on 5-Am paraffin sections using the indirect streptavidin-biotin-peroxidase protocol (19). Sections were incubated with the primary antibody at 1:100 dilution, at 4jC overnight. Haematoxylin was used as counterstain. N, normal tissue; T, tumor. Hyp and Dys, hyperplastic and dysplastic lesions, respectively. B, immunohistochemical analysis of Ser536-phosphorylated p65 (pp65) and p53 on serial sections of a head and neck squamous cell carcinomas harboring hyperplastic and tumor (T) tissue in the same section (middle). Direct DNA sequencing identified the p53 mutation in this tumor as Val175Phe (data not shown). Staining was essentially as in (A), except that a polyclonal anti-phospho-p65 NFnB (Ser536) antibody (Cell Signaling Technology, Danvers, MA) was employed to monitor NFnB activation. Sections were incubated with the primary antibody at 1:10 (phospho-p65) or 1:100 dilution (p53) at 4jC overnight. Higher magnification images of the dashed boxes are also shown. Arrows indicate areas of higher and lower phospho-p65 staining intensity within the tumor. Greater detail is provided in Supplementary Fig. S2.

Article Snippet: Staining was essentially as in (A ), except that a polyclonal anti-phospho-p65 NFnB (Ser536) antibody (Cell Signaling Technology, Danvers, MA) was employed to monitor NFnB activation.

Techniques: Mutagenesis, Over Expression, Activation Assay, Immunohistochemical staining, Staining, Derivative Assay, Formalin-fixed Paraffin-Embedded, Sequencing, Immunohistochemistry, Generated, Bioprocessing, Incubation, DNA Sequencing

Journal: iScience

Article Title: Regulation of Il6 expression by single CpG methylation in downstream of Il6 transcription initiation site

doi: 10.1016/j.isci.2022.104118

Figure Lengend Snippet:

Article Snippet: Transferred membranes were applied for blocking with 5% skim milk in TBST buffer (0.5 M Tris, 1.38 M NaCl, 0.027 M KCl, 0.05% Tween 20) and then were incubated with anti-phospho-IRF3 (Ser396) mouse antibody (4D4G; Cell Signaling Technology), anti-IRF3 mouse antibody (D83B9; Cell Signaling Technology), anti-phosphor-p65 (Ser536) mouse antibody (93H1; Cell Signaling Technology), or anti-p65 mouse antibody (D14E12; Cell Signaling Technology).

Techniques: Virus, Recombinant, Cloning, Ligation, DNA Methylation Assay, RNA Extraction, Plasmid Preparation, Transfection, Derivative Assay, Software, Binding Assay, CRISPR

Journal: International Journal of Molecular Medicine

Article Title: Epigenetic regulation of COX-2 expression by DNA hypomethylation via NF-κB activation in ketamine-induced ulcerative cystitis

doi: 10.3892/ijmm.2019.4252

Figure Lengend Snippet: Ketamine treatment induced the translocation of NF-κB p65 to activate COX-2 expression and PGE2 production. The COX-2 and NF-κB p65 expression of bladder tissue was measured by (A) western blot analysis and (B) quantified against β-actin or Lamin A/C. Results were normalized as the control group=100%. (C) An ELISA was applied to assess the level of PGE2 production for COX-2 enzyme activity in the bladder. Values are presented as the mean ± standard deviation for n=6. * P<0.05 and ** P<0.01 vs. the control group. †† P<0.01 vs. the ketamine group. Double immunostaining of COX-2 (red, left panels) and NF-κB p65 (green, middle panels) for bladder is shown in the (D) control group, the (E) ketamine group and the (F) ketamine+COX-2 inhibitor group. Nuclear DNA was labeled with DAPI (blue). The merged image from left and middle panels (yellow, right panels) is shown. Most COX-2 staining co-labeled with NF-κB p65 (arrows) in the UL and SL was identified in the ketamine group. Scale bar=100 µ m. SL, suburothelial layer; UL, urothelial layer; COX, cyclooxygenase; NF, nuclear factor; PGE, prostaglandin.

Article Snippet: ChIP was performed with primary antibodies for anti-Histone H3K4 (tri methyl K4; cat. no. AB8580; Abcam; rabbit polyclonal IgG; 1:100), H3K9 (cat. no. AB8898; Abcam; rabbit polyclonal IgG; 1:100), H3K27 (cat. no. AB6002; Abcam; mouse monoclonal IgG; 1:100), H3K36 (cat. no. AB9050; Abcam; rabbit polyclonal IgG; 1:100) and H3K79 (cat. no. AB2621; Abcam; rabbit poly-clonal IgG; 1:100), NF-κB p65 (cat. no. 6956S; Cell Signaling Technology, Inc.; mouse monoclonal IgG2b; 1:100), and IgG (cat. no. 6180-01; negative control; SouthernBiotech; rabbit IgG; 1:500) overnight.

Techniques: Translocation Assay, Expressing, Western Blot, Control, Enzyme-linked Immunosorbent Assay, Activity Assay, Standard Deviation, Double Immunostaining, Labeling, Staining

Journal: International Journal of Molecular Medicine

Article Title: Epigenetic regulation of COX-2 expression by DNA hypomethylation via NF-κB activation in ketamine-induced ulcerative cystitis

doi: 10.3892/ijmm.2019.4252

Figure Lengend Snippet: Methylation status of the CpG site in the COX-2 promoter region in ketamine-induced ulcerative cystitis. (A) Schematic diagram of COX-2 promoter spanning the region from -1,522 to -71 bp with respect to the ATG start site (+1) (translation initiation site) and targeted sites for methylation-specific PCR region and bisulfite genomic sequencing analysis are shown. Additionally, five putative NF-κB DNA binding sites in the region were identified by TFBIND software ( http://tfbind.hgc.jp/ ). The broad arrow indicated the direction of transcription. A total of five putative NF-κB binding sequences for methylation analysis are denoted. (B) The bisulfite-COX-2 sequence in the promoter had a size of 1,452 bp and contained 44 CpG sites as identified by nucleotide sequence analysis for potential methylation ( http://www.genome.ucsc.edu ). There were 10 CpG sites in the region I fragment of COX-2 promoter ranging from −299 to −71 bp, 10 CpG sites in the region II ranging from −551 to −286 bp, 11 CpG sites in the region III ranging from -830 to -548 bp, 6 CpG sites in the region IV ranging from -1,195 to -829 bp and 7 CpG sites in the region V ranging from -1,522 to -1,181 bp for methylation sequencing analysis. A total of five sets of primers were designed to amplify the bisulfite genomic sequence. (C) Bisulfite sequencing chromatogram of CpG sites in bladder tissue. Each circle denoted as one CpG site and represented the methylation status of the CpGs. The clone was sequenced from PCR products generated from amplification of bisulfite-treated DNA. The percentage of methylation at a single CpG site was calculated from the sequencing results of four independent clones. ○, unmethylated cytosines; •, methylated cytosines. NF, nuclear factor; IL, interleukin; NFAT/NF-IL6, nuclear factor of activated T cells/NF-IL-6; AP-1, activator protein 1; CRE, cAMP-response element; SP-1, specificity protein 1; TATA, TATA box motif; COX, cyclooxygenase.

Article Snippet: ChIP was performed with primary antibodies for anti-Histone H3K4 (tri methyl K4; cat. no. AB8580; Abcam; rabbit polyclonal IgG; 1:100), H3K9 (cat. no. AB8898; Abcam; rabbit polyclonal IgG; 1:100), H3K27 (cat. no. AB6002; Abcam; mouse monoclonal IgG; 1:100), H3K36 (cat. no. AB9050; Abcam; rabbit polyclonal IgG; 1:100) and H3K79 (cat. no. AB2621; Abcam; rabbit poly-clonal IgG; 1:100), NF-κB p65 (cat. no. 6956S; Cell Signaling Technology, Inc.; mouse monoclonal IgG2b; 1:100), and IgG (cat. no. 6180-01; negative control; SouthernBiotech; rabbit IgG; 1:500) overnight.

Techniques: Methylation, Genomic Sequencing, Binding Assay, Software, Sequencing, Methylation Sequencing, Generated, Amplification, Clone Assay

Journal: International Journal of Molecular Medicine

Article Title: Epigenetic regulation of COX-2 expression by DNA hypomethylation via NF-κB activation in ketamine-induced ulcerative cystitis

doi: 10.3892/ijmm.2019.4252

Figure Lengend Snippet: Methylation status of the CpG site in the COX-2 promoter region III in ketamine-induced ulcerative cystitis. (A) A schematic depiction of COX-2 promoter region III and targeted sites for methylation-specific PCR region is presented. Region III sequence spanned from -830 to -548 relative to the transcription start site (+1) and contained 11 CpG dinucleotides. (B) Bisulfite sequencing data for region III of rat COX-2 promoter were analyzed. This promoter fragment contained 11 CpG sites that were marked with a red color and number. The potential site for NF-κB binding was marked with black square box and bisulfite methylated NFκB3 sequence was located at −824 to −814 bp. Bisulfite genomic sequencing traces were obtained from the PCR products from bisulfite-treated DNA using primers indicated in blue font, including forward and reverse. (C) The trace represented an approximation of the 'average' methylation status at each CpG residue. CpG sites are highlighted as black arrows. Sample number (n)=6. COX, cyclooxygenase; NF, nuclear factor.

Article Snippet: ChIP was performed with primary antibodies for anti-Histone H3K4 (tri methyl K4; cat. no. AB8580; Abcam; rabbit polyclonal IgG; 1:100), H3K9 (cat. no. AB8898; Abcam; rabbit polyclonal IgG; 1:100), H3K27 (cat. no. AB6002; Abcam; mouse monoclonal IgG; 1:100), H3K36 (cat. no. AB9050; Abcam; rabbit polyclonal IgG; 1:100) and H3K79 (cat. no. AB2621; Abcam; rabbit poly-clonal IgG; 1:100), NF-κB p65 (cat. no. 6956S; Cell Signaling Technology, Inc.; mouse monoclonal IgG2b; 1:100), and IgG (cat. no. 6180-01; negative control; SouthernBiotech; rabbit IgG; 1:500) overnight.

Techniques: Methylation, Sequencing, Methylation Sequencing, Binding Assay, Genomic Sequencing, Residue

Journal: International Journal of Molecular Medicine

Article Title: Epigenetic regulation of COX-2 expression by DNA hypomethylation via NF-κB activation in ketamine-induced ulcerative cystitis

doi: 10.3892/ijmm.2019.4252

Figure Lengend Snippet: Methylation status of the CpG site in COX-2 promoter region ranging from −1,181 to −1,522 in ketamine-induced ulcerative cystitis. (A) A schematic depiction of region V and sequence ranging from -1,181 to −1,522 relative to the transcription start site (+1) is presented. (B) Bisulfite genomic sequencing analysis for region V in the COX-2 promoter. Genomic sequencing of bisulfite-COX-2 region V-associated 7 CpG sites was obtained from the PCR products from bisulfite-treated DNA using primers (blue color). The site-specific methylation of the CpG sites (dinucleotides) at −1,442, −1,432 and −1,411 bp located the sequence of NF-κB binding sites in the COX- promoter regions ranging from -1,442 to -1,404 bp. The potential site for NF-κB binding was marked with black square box and the genomic NFκB4 was located at -1,413 to -1,404 bp, 5′-GTC GAT TCC C-3′ (Bisulfite sequence of mNFκB4: 5′-GTC GAT TTT T-3′) and NFκB5 located at -1,442 to -1,432 bp, 5′-CGG TAG TTT CC-3′ (Bisulfite sequence of mNFκB5: 5′-CGG TAG TTT TC-3′). (C) Methylation patterns of the bisulfite-COX-2 region V in three groups. CpG sites were highlighted as arrows. (D) The methylation level of region V in the COX-2 promoter. The methylated percentage of each individual CpG site from bladder tissue was presented. (E) The percentage of total seven CpG sites methylation in region IV. The methylation level of region V from the control group showed a 3.11-fold increase compared with in the ketamine group and a 1.40-fold increase compared with in the ketamine+COX-2 group. Values were the mean ± standard deviation for n=6. * P<0.05 and ** P<0.01 vs. the control group. † P<0.05 and †† P<0.01 vs. the ketamine group. COX, cyclooxygenase; NF-κB4, nuclear factor-κB binding site.

Article Snippet: ChIP was performed with primary antibodies for anti-Histone H3K4 (tri methyl K4; cat. no. AB8580; Abcam; rabbit polyclonal IgG; 1:100), H3K9 (cat. no. AB8898; Abcam; rabbit polyclonal IgG; 1:100), H3K27 (cat. no. AB6002; Abcam; mouse monoclonal IgG; 1:100), H3K36 (cat. no. AB9050; Abcam; rabbit polyclonal IgG; 1:100) and H3K79 (cat. no. AB2621; Abcam; rabbit poly-clonal IgG; 1:100), NF-κB p65 (cat. no. 6956S; Cell Signaling Technology, Inc.; mouse monoclonal IgG2b; 1:100), and IgG (cat. no. 6180-01; negative control; SouthernBiotech; rabbit IgG; 1:500) overnight.

Techniques: Methylation, Sequencing, Genomic Sequencing, Binding Assay, Control, Standard Deviation

Journal: International Journal of Molecular Medicine

Article Title: Epigenetic regulation of COX-2 expression by DNA hypomethylation via NF-κB activation in ketamine-induced ulcerative cystitis

doi: 10.3892/ijmm.2019.4252

Figure Lengend Snippet: Analyses of NF-κB binding sequences and DNA methylation-associated enzymes responsible for the promoter activity of COX-2 gene. (A) Schematic diagram of the COX-2 promoter and targeted sites ranging from -1,522 to -71 bp for ChIP analysis was presented. A total of five potential NF-κB binding sequences in the COX-2 promoter are denoted in the diagram. DNA fragments from ChIP assay were amplified using primer sets to amplify the sequence of the ChIP product. A total of four sets of primers were denoted as primer I-IV, which included five NF-κB binding sequences. ChIP analysis of the potential NF-κB binding sequence in the COX-2 promoter is shown. Formaldehyde cross-linked protein chromatin complexes were immunoprecipitated and genomic DNA was analyzed by (B) PCR and (D) qPCR using primers that recognized the COX-2 promoter region. Input for each reaction was used for internal control of samples loading. IgG was employed as negative control. ChIP analysis of histone H3 methylation on the COX-2 promoter was analyzed by (C) PCR and (E) qPCR. ChIP assay was also applied to anti-tri methyl histone H3 (H3K4m3, H3K9m3, H3K27m3, H3K36m3 and H3K79m3) specific for histone H3 tri-methylated at Lysine (K4, K9, K27, K36, and K79). These antibodies were used to provide direct evidence that histone H3 modulated COX-2 transcriptional regulation by modulating methylation of the COX-2 promoter region ranging from -1,522 to -1,331 bp. Results were normalized as the control group=100%. The mRNA levels of DNMTs (DNMT1, 3a and 3b) for evaluation of the DNA methylation (F) and TETs (TET1-3) for evaluation of the DNA demethylation (G) by reverse transcription-qPCR. The level of TETs expression in the ketamine group was represented as the number of fold increases compared with the control group. Data were expressed as the mean ± standard deviation for n=8. * P<0.05 and ** P<0.01 vs. the control group. †† P<0.01 vs. the ketamine group. ChIP, chromatin immunoprecipitation; TET, Ten-Eleven-Translocation; DNMT, DNA methyltransferase; COX, cyclooxygenase; q, quantitative.

Article Snippet: ChIP was performed with primary antibodies for anti-Histone H3K4 (tri methyl K4; cat. no. AB8580; Abcam; rabbit polyclonal IgG; 1:100), H3K9 (cat. no. AB8898; Abcam; rabbit polyclonal IgG; 1:100), H3K27 (cat. no. AB6002; Abcam; mouse monoclonal IgG; 1:100), H3K36 (cat. no. AB9050; Abcam; rabbit polyclonal IgG; 1:100) and H3K79 (cat. no. AB2621; Abcam; rabbit poly-clonal IgG; 1:100), NF-κB p65 (cat. no. 6956S; Cell Signaling Technology, Inc.; mouse monoclonal IgG2b; 1:100), and IgG (cat. no. 6180-01; negative control; SouthernBiotech; rabbit IgG; 1:500) overnight.

Techniques: Binding Assay, DNA Methylation Assay, Activity Assay, Amplification, Sequencing, Immunoprecipitation, Control, Negative Control, Methylation, Reverse Transcription, Expressing, Standard Deviation, Chromatin Immunoprecipitation, Translocation Assay

Journal: International Journal of Molecular Medicine

Article Title: Epigenetic regulation of COX-2 expression by DNA hypomethylation via NF-κB activation in ketamine-induced ulcerative cystitis

doi: 10.3892/ijmm.2019.4252

Figure Lengend Snippet: A model illustrating ketamine treatment promoting the hypomethylation of COX-2 gene promoter via NF-κB in ketamine-induced ulcerative cystitis of rat bladder. Inflammatory stimuli of ketamine induced nuclear translocation of NF-κB and increased nuclear NF-κB level. The activated nuclear NF-κB bound to specific sequences of COX-2 promoter and thereafter enhanced hypomethylation of COX-2 gene promoter. The hypomethylation of COX-2 promoter increased COX-2 expression and the level of PGE2 production. PGE, prostaglandin; IKK, IκB kinase; P, phosphorylation; NFAT/NF-IL6, nuclear factor of activated T cells/NF-IL-6; AP-1, activator protein 1; CRE, cAMP-response element; SP-1, specificity protein 1; TATA, TATA box motif.

Article Snippet: ChIP was performed with primary antibodies for anti-Histone H3K4 (tri methyl K4; cat. no. AB8580; Abcam; rabbit polyclonal IgG; 1:100), H3K9 (cat. no. AB8898; Abcam; rabbit polyclonal IgG; 1:100), H3K27 (cat. no. AB6002; Abcam; mouse monoclonal IgG; 1:100), H3K36 (cat. no. AB9050; Abcam; rabbit polyclonal IgG; 1:100) and H3K79 (cat. no. AB2621; Abcam; rabbit poly-clonal IgG; 1:100), NF-κB p65 (cat. no. 6956S; Cell Signaling Technology, Inc.; mouse monoclonal IgG2b; 1:100), and IgG (cat. no. 6180-01; negative control; SouthernBiotech; rabbit IgG; 1:500) overnight.

Techniques: Translocation Assay, Expressing, Phospho-proteomics