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  • 99
    Cell Signaling Technology Inc p65
    Confirmation of PDGFRβ-specific effects in Rb cells by PDGFRB siRNA. PDGFRB gene expression in Y79 was targeted using commercially available siRNA. (A) qPCR analyses confirmed effective knockdown of PDGFRB mRNA in siRNA-transfected samples compared to nonspecific oligonucleotides (scramble) and untreated cells. (B–D) The percentages of viable and apoptotic cells were determined by flow cytometry analysis using (B) the annexin V − PI − phenotype for live cells; *P = 0.019 (C) the percentage of early apoptotic (annexin V + ); *P = 0.0107; **P = 0.0052 and (D) late apoptotic (annexin V + PI + ) cells; *P = 0.0120; **P = 0.0029. (E) Western blot analyses were done to measure activity levels of MDM2 after transfection of PDGFRB siRNA. (F–H) Using the Amnis FlowSight Imaging Cytometer, untreated, scramble, or PDGFRB siRNA-transfected Y79 cells were labeled and analyzed for (F) the percentage of <t>p65</t> nuclear translocation; ***P
    P65, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 5452 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc nf κb p65
    Osthole sensitization of radiation in inhibition of cervical cancer cell viability by targeting multiple signaling pathways. Cervical cancer cells were grown and treated with osthole in combination with radiation. Combination treatment suppressed the protein expression of vimentin, N-cadherin, MMP-2 and MMP-9, induced the cleavage of pro-apoptotic caspase, inhibited the phosphorylation of γH2AX, IKKα and <t>p65</t> proteins by ATM, and promoted the translocation of NF-κB from cell nuclei to cytoplasm. IR, irradiation; NF-κB, nuclear factor-κB; IKKα, inhibitor of NF-κB kinase α; p-, phosphorylated; ATM, ataxia telangiectasia mutated; MMP, matrix metalloproteinase.
    Nf κb P65, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 5030 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology p65
    cAMP-PKA pathway induces PTEN expression, but <t>p65</t> blocks this induction. NIH 3T3 cells were transfected with either PTEN promoter-Luc reporter construct or CRE (cAMP response element)-Luc reporter construct and left untreated or treated with forskolin (FK) plus IBMX or 8-bromo-cAMP for 24 h before harvesting the cells. Luciferase activity was determined at 48 h posttransfection (A). NIH 3T3 cells were treated with 8-bromo-cAMP for the indicated time periods, and whole-cell lysates were subjected to Western blot analysis for PTEN or actin (B). NIH 3T3 cells were cotransfected with the PTEN promoter-Luc reporter construct along with p65 or 276A mutant of p65, and 8-bromo-cAMP was added 6 h before harvesting the cells. Luciferase activity was determined at 48 h posttransfection (C). Relative Luc activity, normalized to the corresponding β-galactosidase activity, is shown.
    P65, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 5928 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Santa Cruz Biotechnology nf κb p65
    NEK2 activates NF-κB signaling via PP1α/AKT. ( A ) USP7 was knocked down in ARP1 cells transduced with NEK2-OE after 72 hours induction with doxycycline (DOX). Nuclear and cytosolic fractionations were carried out. <t>p65</t> levels were analyzed between EV and NEK2-OE with or without USP7 shRNA by Western blot. β-Actin and histone H3 (H3) were used as cytosolic and nuclear markers, respectively. ( B – D ) EV and NEK2-OE ARP1, OCI-MY5, and H1299 cells were lysed. NEK2, p65-S536 phosphorylation, IKK phosphorylation, and IκBα were analyzed by Western blot. ( E ) H1299 cells transiently transfected with EV or NEK2-OE (WT) or NEK2-K37R mutant (NEK2-Dead) were lysed, and NEK2 and p65-S536 phosphorylation was analyzed by Western blot. ( F ) ARP1 and OCI-MY5 cells transfected with EV or NEK2-OE were treated with vehicle or MK-2206 2HCl, an AKT inhibitor, for 30 minutes and then cells were lysed. p65-S536 phosphorylation was analyzed by Western blot. ( G ) NEK2-shRNA ARP1 cells were induced with DOX for 48 hours and then treated with tautomycin, a PP1α inhibitor, for another 24 hours. NEK2, p-p65-S536, p-PP1α, and p-AKT were analyzed by Western blot.
    Nf κb P65, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 94/100, based on 3736 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc p p65
    NEK2 activates NF-κB signaling via PP1α/AKT. ( A ) USP7 was knocked down in ARP1 cells transduced with NEK2-OE after 72 hours induction with doxycycline (DOX). Nuclear and cytosolic fractionations were carried out. <t>p65</t> levels were analyzed between EV and NEK2-OE with or without USP7 shRNA by Western blot. β-Actin and histone H3 (H3) were used as cytosolic and nuclear markers, respectively. ( B – D ) EV and NEK2-OE ARP1, OCI-MY5, and H1299 cells were lysed. NEK2, p65-S536 phosphorylation, IKK phosphorylation, and IκBα were analyzed by Western blot. ( E ) H1299 cells transiently transfected with EV or NEK2-OE (WT) or NEK2-K37R mutant (NEK2-Dead) were lysed, and NEK2 and p65-S536 phosphorylation was analyzed by Western blot. ( F ) ARP1 and OCI-MY5 cells transfected with EV or NEK2-OE were treated with vehicle or MK-2206 2HCl, an AKT inhibitor, for 30 minutes and then cells were lysed. p65-S536 phosphorylation was analyzed by Western blot. ( G ) NEK2-shRNA ARP1 cells were induced with DOX for 48 hours and then treated with tautomycin, a PP1α inhibitor, for another 24 hours. NEK2, p-p65-S536, p-PP1α, and p-AKT were analyzed by Western blot.
    P P65, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 2726 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology anti p65
    Quantification of IL‐1α‐mediated enhancer modifications and <t>p65</t> NF‐κB binding in the human IL8 and CXCL2 chemokine loci Published ChIP‐seq data from KB cells (Jurida et al , 2015 ; GSE64224 and GSE52470) were used to annotate active enhancers and p65 NF‐κB recruitment in untreated cells compared to cells stimulated with IL‐1α for 60 min ± TAKi. The browser views show ChIP‐seq profiles for all conditions of the IL8 and CXCL2 chemokine loci. Gray areas highlight four chromatin regions with IL‐1α‐inducible H3K27 acetylation and p65 binding. As indicated by black horizontal bars, these chromatin regions were provisionally designated by us as “class II enhancers” (Jurida et al , 2015 ) to distinguish them from the entire repertoire of all active (i.e., H3K4me1‐ and H3K27ac‐positive) enhancers. Vertical bars indicate predicted NF‐κB motifs in the DNA sequence and the positions of all sgRNAs used in this study for CRISPRa (see Fig 6 ) or for enhancer or promoter deletions (see Figs 3 and EV4). Bar graphs show cumulative read counts across the four IL‐1α‐regulated and TAKi‐sensitive class II enhancers. The two flanking enhancers with strongest p65 binding (2 and 4) are the ones investigated in detail in this study.
    Anti P65, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 3001 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc phospho p65
    Fargesin blocked the activation of NF-κB in vitro. ( A ) RAW264.7 cells were treated as described in the Methods and <t>p-p65</t> localization was visualized under a fluorescence microscope. Scale bar corresponds to 200 μm. ( B ) RAW264.7 cells were treated as described in the Methods. NF-κB promoter-driven luciferase activity was determined using a dual luciferase assay system, and values were expressed as the fold induction of the control cells. Data were expressed as mean ± SD of quadruplicates of two independent experiments. *** p
    Phospho P65, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1792 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc anti nf κb p65
    Fargesin blocked the activation of NF-κB in vitro. ( A ) RAW264.7 cells were treated as described in the Methods and <t>p-p65</t> localization was visualized under a fluorescence microscope. Scale bar corresponds to 200 μm. ( B ) RAW264.7 cells were treated as described in the Methods. NF-κB promoter-driven luciferase activity was determined using a dual luciferase assay system, and values were expressed as the fold induction of the control cells. Data were expressed as mean ± SD of quadruplicates of two independent experiments. *** p
    Anti Nf κb P65, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1352 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Cell Signaling Technology Inc phospho nf κb p65
    Inhibitory effects of cecropin-TY1 on LPS-stimulated inflammatory response signal pathway in peritoneal macrophages. a – b Effects of cecropin-TY1 on ( a ) MAPKs and ( b ) NF-κB activation in peritoneal macrophages after 100 ng/mL LPS stimulation. The photograph is a representative one of three independent experiments. Peritoneal macrophages were stimulated with or without LPS (100 ng/mL), then different concentrations of cecropin-TY1 (cec-TY1, 5, 10, 20 μg/mL) were added immediately and incubated for 30 min, and macrophages were lysed for western blot analysis. ( c – i Ratio of ( c ) P-ERK1 (42 kDa), ( d ) P-ERK2 (44 kDa), ( e ) P-JNK1 (46 kDa), ( f ) P-JNK2 (54 kDa), ( g ) P-p38, ( h ) P-IκBα and ( i ) <t>P-p65</t> to β-actin. Band densities were analyzed using Quantity One software (Bio-Rad, Richmond, CA USA). Data were presented as mean ± SEM. * P
    Phospho Nf κb P65, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1409 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Santa Cruz Biotechnology anti nf κb p65
    Quantification of IL‐1α‐mediated enhancer modifications and <t>p65</t> NF‐κB binding in the human IL8 and CXCL2 chemokine loci Published ChIP‐seq data from KB cells (Jurida et al , 2015 ; GSE64224 and GSE52470) were used to annotate active enhancers and p65 NF‐κB recruitment in untreated cells compared to cells stimulated with IL‐1α for 60 min ± TAKi. The browser views show ChIP‐seq profiles for all conditions of the IL8 and CXCL2 chemokine loci. Gray areas highlight four chromatin regions with IL‐1α‐inducible H3K27 acetylation and p65 binding. As indicated by black horizontal bars, these chromatin regions were provisionally designated by us as “class II enhancers” (Jurida et al , 2015 ) to distinguish them from the entire repertoire of all active (i.e., H3K4me1‐ and H3K27ac‐positive) enhancers. Vertical bars indicate predicted NF‐κB motifs in the DNA sequence and the positions of all sgRNAs used in this study for CRISPRa (see Fig 6 ) or for enhancer or promoter deletions (see Figs 3 and EV4). Bar graphs show cumulative read counts across the four IL‐1α‐regulated and TAKi‐sensitive class II enhancers. The two flanking enhancers with strongest p65 binding (2 and 4) are the ones investigated in detail in this study.
    Anti Nf κb P65, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1438 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p65  (Abcam)
    99
    Abcam p65
    Effect of the reactive oxygen species (ROS) inhibitor N-acetylcysteine (NAC) on the nuclear factor (NF)-κB signaling pathway in xanthohumol (Xn)-treated AGS cells. Cells were pre-treated with the ROS inhibitor NAC (5 mM) for 1 h, and then treated with Xn (20 µ M) for 24 h; they were then harvested and lysed to measure NF-κB signaling proteins through western blotting. (A-C) Expression of IκBα and p-IκBα protein; (D-F) Expression of nuclear and cytosolic <t>p65</t> protein. Histone H3 served as the nuclear loading control, GAPDH served as the cytosolic loading control. Data are expressed as mean ± standard error of the mean. n=3. **P
    P65, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 1166 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam nf κb p65
    12-HETE promotes the activation and nuclear translocation of NF-κB through the ILK pathway. Notes: ( A ) 1 µM 12-HETE treatment led to increased levels of phosphorylated NF-κB <t>p65</t> but was antagonized by the knockdown of ILK. ( B ) 1 µM 12-HETE significantly increased the protein level of nuclear NF-κB p65 and decreased the protein level of NF-κB p65 in the cytoplasm via ILK. ( C ) The 12-HETE-induced phosphorylation of IKBa was mediated by ILK in ovarian cancer cells. All values are represented as the mean±SEM from three or more independent batches of cells (* P
    Nf κb P65, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 1278 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc p nf κb p65
    Inhibitory effects of hypericin-photodynamic therapy on NF-κB activation in MH7A cells. Cells were treated with HYP (0–4 μM) and PDT. NF-κB pathway proteins and mRNA were measured with Western blot and qRT-PCR, with β-actin used as an internal control. (A) Western blot data is shown for p-NF-κB <t>p65,</t> NF-κB p65 and p-IκBα and densitometric analysis performed using ImageJ software. Relative quantities of p-NF-κB p65 (B), NF-κB p65 (C) and p-IκBα (D) were normalized to untreated cells (0 μM HYP; 100%). (E) NF-κB p65 mRNA was assessed using qRT-PCR. Gene expression was normalized to GAPDH. Data are means±SD (n=3); *** P
    P Nf κb P65, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 1039 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Abcam anti nf kb p65 antibody
    Synergistic inhibitory effects of AC and the NF-κB signaling pathway inhibitor JSH-23 on colorectal cancer cells. (A) CCK-8 assay was performed to detect the viability of HCT116 cells treated with AC and JSH-23 for 24, 48 and 72 h. (B) Cell cycle of HCT116 cells treated with AC and JSH-23 for 24 h was assessed using flow cytometry. (C) Statistical analysis of cell cycle of HCT116 cells co-treated with AC and JSH-23 for 24 h. (D) Cell cycle of HCT116 cells co-treated with AC and JSH-23 for 72 h was measured by flow cytometry. (E) Quantification of cell cycle of HCT116 cells co-treated with AC and JSH-23 for 72 h. (F) Cell apoptosis of HCT116 cells co-treated with AC and JSH-23 for 24 h was determined using flow cytometry. (G) Cell apoptosis of HCT116 cells co-treated with AC and JSH-23 for 72 h was detected by flow cytometry. (H) Statistical analysis of cell apoptosis of HCT116 cells co-treated with AC and JSH-23 for 24 h. (I) Statistical analysis of cell apoptosis of HCT116 cells co-treated with AC and JSH-23 for 72 h. (J) The phosphorylation of IκBα and <t>P65</t> was measured by western blotting. Data are expressed as mean ± standard deviation from three independent experiments. * P
    Anti Nf Kb P65 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 391 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti phospho p65
    Synergistic inhibitory effects of AC and the NF-κB signaling pathway inhibitor JSH-23 on colorectal cancer cells. (A) CCK-8 assay was performed to detect the viability of HCT116 cells treated with AC and JSH-23 for 24, 48 and 72 h. (B) Cell cycle of HCT116 cells treated with AC and JSH-23 for 24 h was assessed using flow cytometry. (C) Statistical analysis of cell cycle of HCT116 cells co-treated with AC and JSH-23 for 24 h. (D) Cell cycle of HCT116 cells co-treated with AC and JSH-23 for 72 h was measured by flow cytometry. (E) Quantification of cell cycle of HCT116 cells co-treated with AC and JSH-23 for 72 h. (F) Cell apoptosis of HCT116 cells co-treated with AC and JSH-23 for 24 h was determined using flow cytometry. (G) Cell apoptosis of HCT116 cells co-treated with AC and JSH-23 for 72 h was detected by flow cytometry. (H) Statistical analysis of cell apoptosis of HCT116 cells co-treated with AC and JSH-23 for 24 h. (I) Statistical analysis of cell apoptosis of HCT116 cells co-treated with AC and JSH-23 for 72 h. (J) The phosphorylation of IκBα and <t>P65</t> was measured by western blotting. Data are expressed as mean ± standard deviation from three independent experiments. * P
    Anti Phospho P65, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 810 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cell Signaling Technology Inc anti phospho nf κb p65
    Proteasome inhibitors act directly on the HIV LTR to activate HIV in an NF-κB-dependent manner. (A) Chronically HIV-infected J-Lat 10.6 cells were treated with DMSO, bortezomib, or ixazomib and assessed for GFP expression over time. Depicted are means (±SD) of the results of four independent experiments. (B) J-Lat 10.6 cells treated as for panel A were assessed for HIV p55 Gag expression by Western blotting. (C) Jurkat CD4 + T cells transfected with an HIV-1 LTR firefly luciferase reporter construct were treated with control, bortezomib, or ixazomib, and luciferase activity was measured and normalized to that of Renilla luciferase. Depicted is fold luciferase induction relative to that of control-treated cells (means and SD of the results of five independent experiments). (D) Jurkat CD4 + T cells transfected with the HIV LTR luciferase construct with or without deletions in the NF-κB-binding site (ΔKb) were treated with control, bortezomib, or ixazomib and assessed for luciferase activity, normalized to that of Renilla luciferase. Depicted is fold luciferase induction relative to that of control-treated cells (means and SD of the results of two independent experiments). (E) J-Lat 10.6 cells were treated with control, bortezomib, or ixazomib for 6 or 24 h, as indicated, and expression of NF-κB <t>p65,</t> Ser536 phospho-NF-κB, IκBα, and Ser32 phospho-IκBα was assessed by Western blot analysis. *, P
    Anti Phospho Nf κb P65, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 99/100, based on 519 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti p65
    <t>NF-κB/p65</t> is responsible for the downregulation of MMP-9 and Bfl-1 mediated by fordin in U-2 OS and HepG2 cells. The expression of p65 (phosphorylated and total), MMP-9 and Bfl-1 were determined by western blot analysis in the various treated cells and normalized to β-actin or Histone H3. (A) U-2 OS and (B) HepG2 cells were incubated with various concentrations of fordin for 24 h; * P
    Anti P65, supplied by Abcam, used in various techniques. Bioz Stars score: 97/100, based on 568 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam anti nf kb p65 phospho s536 antibody
    <t>NF-κB/p65</t> is responsible for the downregulation of MMP-9 and Bfl-1 mediated by fordin in U-2 OS and HepG2 cells. The expression of p65 (phosphorylated and total), MMP-9 and Bfl-1 were determined by western blot analysis in the various treated cells and normalized to β-actin or Histone H3. (A) U-2 OS and (B) HepG2 cells were incubated with various concentrations of fordin for 24 h; * P
    Anti Nf Kb P65 Phospho S536 Antibody, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 255 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Confirmation of PDGFRβ-specific effects in Rb cells by PDGFRB siRNA. PDGFRB gene expression in Y79 was targeted using commercially available siRNA. (A) qPCR analyses confirmed effective knockdown of PDGFRB mRNA in siRNA-transfected samples compared to nonspecific oligonucleotides (scramble) and untreated cells. (B–D) The percentages of viable and apoptotic cells were determined by flow cytometry analysis using (B) the annexin V − PI − phenotype for live cells; *P = 0.019 (C) the percentage of early apoptotic (annexin V + ); *P = 0.0107; **P = 0.0052 and (D) late apoptotic (annexin V + PI + ) cells; *P = 0.0120; **P = 0.0029. (E) Western blot analyses were done to measure activity levels of MDM2 after transfection of PDGFRB siRNA. (F–H) Using the Amnis FlowSight Imaging Cytometer, untreated, scramble, or PDGFRB siRNA-transfected Y79 cells were labeled and analyzed for (F) the percentage of p65 nuclear translocation; ***P

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Targeting the Platelet-Derived Growth Factor-beta Stimulatory Circuitry to Control Retinoblastoma Seeds

    doi: 10.1167/iovs.18-24359

    Figure Lengend Snippet: Confirmation of PDGFRβ-specific effects in Rb cells by PDGFRB siRNA. PDGFRB gene expression in Y79 was targeted using commercially available siRNA. (A) qPCR analyses confirmed effective knockdown of PDGFRB mRNA in siRNA-transfected samples compared to nonspecific oligonucleotides (scramble) and untreated cells. (B–D) The percentages of viable and apoptotic cells were determined by flow cytometry analysis using (B) the annexin V − PI − phenotype for live cells; *P = 0.019 (C) the percentage of early apoptotic (annexin V + ); *P = 0.0107; **P = 0.0052 and (D) late apoptotic (annexin V + PI + ) cells; *P = 0.0120; **P = 0.0029. (E) Western blot analyses were done to measure activity levels of MDM2 after transfection of PDGFRB siRNA. (F–H) Using the Amnis FlowSight Imaging Cytometer, untreated, scramble, or PDGFRB siRNA-transfected Y79 cells were labeled and analyzed for (F) the percentage of p65 nuclear translocation; ***P

    Article Snippet: Samples were blocked with PBS/1% fetal bovine serum before addition of anti-p65 (catalog no. 8242; CST; 1:100) antibody for 1 hour on ice.

    Techniques: Expressing, Real-time Polymerase Chain Reaction, Transfection, Flow Cytometry, Cytometry, Western Blot, Activity Assay, Imaging, Labeling, Translocation Assay

    PDGF-PDGFRβ signaling regulates key downstream signaling mediators. qPCR analyses on mRNA levels of (A) MDM2 and (B) MDM4 relative to untreated Y79 cells under the cell culture conditions described above. HPRT1 used as housekeeping gene. Western blot analyses (C) of MDM2 activity and (D) AKT activity after disruption of the PDGFRβ signaling cascade. IM, *P = 0.0275; rhPDGF + IM, **P = 0.0011; AKT, *P = 0.0054. Evaluation of the antiapoptotic mediator (E) BCL-2 by Western blot (**P = 0.001 and ***P = 0.0002); and the proapoptotic cleaved (active) capsase-3 by ELISA (F), *P = 0.007. (G–L) Downregulation of the VEGFR signaling when PDGFRβ is inhibited. (G) VEGFA mRNA levels were measured by qPCR analysis in both Y79 cells and (H) PDX samples. (I) Both VEGFA and (J) VEGFR2 mRNA levels were measured across respective treatments as well as (K) Western blot analyses of VEGFR2 activity; *P = 0.0191. (L) Measurement of VEGFA levels in a cohort of vitreous samples from healthy controls compared to vitreous of Rb patients. ****P = 0.0001. (M–O) Assessment of the NF-κB signaling. Representing images of Y79 cells (M) labeled and analyzed for nuclear (labeled with DRAQ5) translocation of the p65 subunit of NF-κB (AlexaFluor 488 conjugated), ***P

    Journal: Investigative Ophthalmology & Visual Science

    Article Title: Targeting the Platelet-Derived Growth Factor-beta Stimulatory Circuitry to Control Retinoblastoma Seeds

    doi: 10.1167/iovs.18-24359

    Figure Lengend Snippet: PDGF-PDGFRβ signaling regulates key downstream signaling mediators. qPCR analyses on mRNA levels of (A) MDM2 and (B) MDM4 relative to untreated Y79 cells under the cell culture conditions described above. HPRT1 used as housekeeping gene. Western blot analyses (C) of MDM2 activity and (D) AKT activity after disruption of the PDGFRβ signaling cascade. IM, *P = 0.0275; rhPDGF + IM, **P = 0.0011; AKT, *P = 0.0054. Evaluation of the antiapoptotic mediator (E) BCL-2 by Western blot (**P = 0.001 and ***P = 0.0002); and the proapoptotic cleaved (active) capsase-3 by ELISA (F), *P = 0.007. (G–L) Downregulation of the VEGFR signaling when PDGFRβ is inhibited. (G) VEGFA mRNA levels were measured by qPCR analysis in both Y79 cells and (H) PDX samples. (I) Both VEGFA and (J) VEGFR2 mRNA levels were measured across respective treatments as well as (K) Western blot analyses of VEGFR2 activity; *P = 0.0191. (L) Measurement of VEGFA levels in a cohort of vitreous samples from healthy controls compared to vitreous of Rb patients. ****P = 0.0001. (M–O) Assessment of the NF-κB signaling. Representing images of Y79 cells (M) labeled and analyzed for nuclear (labeled with DRAQ5) translocation of the p65 subunit of NF-κB (AlexaFluor 488 conjugated), ***P

    Article Snippet: Samples were blocked with PBS/1% fetal bovine serum before addition of anti-p65 (catalog no. 8242; CST; 1:100) antibody for 1 hour on ice.

    Techniques: Real-time Polymerase Chain Reaction, Cell Culture, Western Blot, Activity Assay, Enzyme-linked Immunosorbent Assay, Labeling, Translocation Assay

    Osthole sensitization of radiation in inhibition of cervical cancer cell viability by targeting multiple signaling pathways. Cervical cancer cells were grown and treated with osthole in combination with radiation. Combination treatment suppressed the protein expression of vimentin, N-cadherin, MMP-2 and MMP-9, induced the cleavage of pro-apoptotic caspase, inhibited the phosphorylation of γH2AX, IKKα and p65 proteins by ATM, and promoted the translocation of NF-κB from cell nuclei to cytoplasm. IR, irradiation; NF-κB, nuclear factor-κB; IKKα, inhibitor of NF-κB kinase α; p-, phosphorylated; ATM, ataxia telangiectasia mutated; MMP, matrix metalloproteinase.

    Journal: Oncology Reports

    Article Title: Osthole enhances antitumor activity and irradiation sensitivity of cervical cancer cells by suppressing ATM/NF-κB signaling

    doi: 10.3892/or.2018.6514

    Figure Lengend Snippet: Osthole sensitization of radiation in inhibition of cervical cancer cell viability by targeting multiple signaling pathways. Cervical cancer cells were grown and treated with osthole in combination with radiation. Combination treatment suppressed the protein expression of vimentin, N-cadherin, MMP-2 and MMP-9, induced the cleavage of pro-apoptotic caspase, inhibited the phosphorylation of γH2AX, IKKα and p65 proteins by ATM, and promoted the translocation of NF-κB from cell nuclei to cytoplasm. IR, irradiation; NF-κB, nuclear factor-κB; IKKα, inhibitor of NF-κB kinase α; p-, phosphorylated; ATM, ataxia telangiectasia mutated; MMP, matrix metalloproteinase.

    Article Snippet: The following day, the membranes were washed with PBS-Tween-20 three times and then incubated with an anti-rabbit or mouse secondary antibody at the room temperature for 2 h. The primary antibodies were rabbit monoclonal antibodies against Bcl-2 (cat. no. 3498), Bax (cat. no. 14796), cleaved caspase-3 (cat. no. 9664), cleaved caspase-9 (cat. no. 20750), vimentin (cat. no. 5741), N-cadherin (cat. no. 13116), E-cadherin (cat. no. 3195), β-catenin (cat. no. 8480), MMP-2 (cat. no. 40994), MMP-9 (cat. no. 13667), Phospho-ATM (Ser1981; cat. no. 13050), ATM (cat. no. 2873), Phospho-Histone H2A.X (Ser139; cat. no. 2577), Histone H2A.X (cat. no. 7631), NF-κB p65 (cat. no. 8242), Phospho-IKKα (Ser176)/IKKβ (Ser177) (cat. no. 2078), IKKα (cat. no. 2682), Phospho-NF-κB p65 (Ser536; cat. no. 3033), NF-κB p65 (cat. no. 8242) and NF-κB1 p105/p50 (cat. no. 12540; all from Cell Signaling Technology) and used at a dilution of 1:1,000, while the secondary antibody was an anti-rabbit IgG SA00001-2 (Proteintech, Wuhan, China) and used at a dilution of 1:5,000.

    Techniques: Inhibition, Expressing, Translocation Assay, Irradiation

    Osthole promotes NF-κB signaling in radiation-induced cervical cancer cell DNA damage. HeLa cells were grown and treated with osthole (50, 100 or 150 µM) for 24 h and exposed to 10 Gy radiation. Cytoplasmic and nuclear proteins of HeLa cells were separately extracted for western blot analysis of key proteins in the NF-κB signaling pathway, including (A) IKKα, p-IKKα, p65, p-p65 and (B) p50. (C) Subcellular localization of p65 in HeLa cells was examined by analysis with a fluorescence microscope (magnification, ×50). All results are expressed as the mean ± standard deviation (*P

    Journal: Oncology Reports

    Article Title: Osthole enhances antitumor activity and irradiation sensitivity of cervical cancer cells by suppressing ATM/NF-κB signaling

    doi: 10.3892/or.2018.6514

    Figure Lengend Snippet: Osthole promotes NF-κB signaling in radiation-induced cervical cancer cell DNA damage. HeLa cells were grown and treated with osthole (50, 100 or 150 µM) for 24 h and exposed to 10 Gy radiation. Cytoplasmic and nuclear proteins of HeLa cells were separately extracted for western blot analysis of key proteins in the NF-κB signaling pathway, including (A) IKKα, p-IKKα, p65, p-p65 and (B) p50. (C) Subcellular localization of p65 in HeLa cells was examined by analysis with a fluorescence microscope (magnification, ×50). All results are expressed as the mean ± standard deviation (*P

    Article Snippet: The following day, the membranes were washed with PBS-Tween-20 three times and then incubated with an anti-rabbit or mouse secondary antibody at the room temperature for 2 h. The primary antibodies were rabbit monoclonal antibodies against Bcl-2 (cat. no. 3498), Bax (cat. no. 14796), cleaved caspase-3 (cat. no. 9664), cleaved caspase-9 (cat. no. 20750), vimentin (cat. no. 5741), N-cadherin (cat. no. 13116), E-cadherin (cat. no. 3195), β-catenin (cat. no. 8480), MMP-2 (cat. no. 40994), MMP-9 (cat. no. 13667), Phospho-ATM (Ser1981; cat. no. 13050), ATM (cat. no. 2873), Phospho-Histone H2A.X (Ser139; cat. no. 2577), Histone H2A.X (cat. no. 7631), NF-κB p65 (cat. no. 8242), Phospho-IKKα (Ser176)/IKKβ (Ser177) (cat. no. 2078), IKKα (cat. no. 2682), Phospho-NF-κB p65 (Ser536; cat. no. 3033), NF-κB p65 (cat. no. 8242) and NF-κB1 p105/p50 (cat. no. 12540; all from Cell Signaling Technology) and used at a dilution of 1:1,000, while the secondary antibody was an anti-rabbit IgG SA00001-2 (Proteintech, Wuhan, China) and used at a dilution of 1:5,000.

    Techniques: Western Blot, Fluorescence, Microscopy, Standard Deviation

    cAMP-PKA pathway induces PTEN expression, but p65 blocks this induction. NIH 3T3 cells were transfected with either PTEN promoter-Luc reporter construct or CRE (cAMP response element)-Luc reporter construct and left untreated or treated with forskolin (FK) plus IBMX or 8-bromo-cAMP for 24 h before harvesting the cells. Luciferase activity was determined at 48 h posttransfection (A). NIH 3T3 cells were treated with 8-bromo-cAMP for the indicated time periods, and whole-cell lysates were subjected to Western blot analysis for PTEN or actin (B). NIH 3T3 cells were cotransfected with the PTEN promoter-Luc reporter construct along with p65 or 276A mutant of p65, and 8-bromo-cAMP was added 6 h before harvesting the cells. Luciferase activity was determined at 48 h posttransfection (C). Relative Luc activity, normalized to the corresponding β-galactosidase activity, is shown.

    Journal: Molecular and Cellular Biology

    Article Title: Suppression of PTEN Expression by NF-?B Prevents Apoptosis

    doi: 10.1128/MCB.24.3.1007-1021.2004

    Figure Lengend Snippet: cAMP-PKA pathway induces PTEN expression, but p65 blocks this induction. NIH 3T3 cells were transfected with either PTEN promoter-Luc reporter construct or CRE (cAMP response element)-Luc reporter construct and left untreated or treated with forskolin (FK) plus IBMX or 8-bromo-cAMP for 24 h before harvesting the cells. Luciferase activity was determined at 48 h posttransfection (A). NIH 3T3 cells were treated with 8-bromo-cAMP for the indicated time periods, and whole-cell lysates were subjected to Western blot analysis for PTEN or actin (B). NIH 3T3 cells were cotransfected with the PTEN promoter-Luc reporter construct along with p65 or 276A mutant of p65, and 8-bromo-cAMP was added 6 h before harvesting the cells. Luciferase activity was determined at 48 h posttransfection (C). Relative Luc activity, normalized to the corresponding β-galactosidase activity, is shown.

    Article Snippet: Mouse monoclonal antibodies for PTEN (A2B1) and IKKβ (H-4) and rabbit polyclonal antibodies for p65 (C-20), p50 (H-119), IκB-α (C-21), Akt1/2 (H-136), X chromosome-linked IAP (XIAP) (H-202), and cIAP1 (H-83) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.).

    Techniques: Expressing, Transfection, Construct, Luciferase, Activity Assay, Western Blot, Mutagenesis

    p65 represses the PTEN promoter independent of its transcriptional activity. Schematic diagram illustrating various deletion and phosphorylation-defective point mutant constructs of p65 used in this study (A). NIH 3T3 cells were cotransfected with vector, p65, or various p65 deletion-point mutant expression constructs, and the PTEN promoter-Luc reporter construct. Luciferase activity was determined at 48 h posttransfection (B). The bottom panel shows Western blot analysis performed on whole-cell lysates from the transfectants (B). Note the expression of p65 (arrow) or Flag-tagged p65 mutants (asterisk, thick arrow for ΔC, which contains amino acids 1 to 337 of p65) and actin (B). Cos-7 cells were cotransfected with vector, IκB-SR, p65, or various p65 deletion-point mutant expression constructs, and the PTEN promoter-Luc reporter construct. Luciferase activity was determined at 48 h posttransfection (C). Relative Luc activity, normalized to the corresponding β-galactosidase activity, is shown.

    Journal: Molecular and Cellular Biology

    Article Title: Suppression of PTEN Expression by NF-?B Prevents Apoptosis

    doi: 10.1128/MCB.24.3.1007-1021.2004

    Figure Lengend Snippet: p65 represses the PTEN promoter independent of its transcriptional activity. Schematic diagram illustrating various deletion and phosphorylation-defective point mutant constructs of p65 used in this study (A). NIH 3T3 cells were cotransfected with vector, p65, or various p65 deletion-point mutant expression constructs, and the PTEN promoter-Luc reporter construct. Luciferase activity was determined at 48 h posttransfection (B). The bottom panel shows Western blot analysis performed on whole-cell lysates from the transfectants (B). Note the expression of p65 (arrow) or Flag-tagged p65 mutants (asterisk, thick arrow for ΔC, which contains amino acids 1 to 337 of p65) and actin (B). Cos-7 cells were cotransfected with vector, IκB-SR, p65, or various p65 deletion-point mutant expression constructs, and the PTEN promoter-Luc reporter construct. Luciferase activity was determined at 48 h posttransfection (C). Relative Luc activity, normalized to the corresponding β-galactosidase activity, is shown.

    Article Snippet: Mouse monoclonal antibodies for PTEN (A2B1) and IKKβ (H-4) and rabbit polyclonal antibodies for p65 (C-20), p50 (H-119), IκB-α (C-21), Akt1/2 (H-136), X chromosome-linked IAP (XIAP) (H-202), and cIAP1 (H-83) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.).

    Techniques: Activity Assay, Mutagenesis, Construct, Plasmid Preparation, Expressing, Luciferase, Western Blot

    NF-κB down-regulates the PTEN promoter. NIH 3T3 cells were cotransfected with pGL3 basic-Luc, PTEN promoter-Luc, or cyclin D1-Luc reporter construct, as indicated, and p65 expression construct or vector as a control along with β-galactosidase expression plasmid. Relative Luc activity, normalized to the corresponding β-galactosidase activity is shown (A). Relative Luc activity of 100 corresponds to 16,000 to 25,000 U of the raw Luc activity values normalized to β-galactosidase activity for all of the luciferase reporter experiments. NIH 3T3 cells were cotransfected with the PTEN promoter-Luc reporter construct or NF-κB-Luc reporter construct, as indicated, and IκB-SR expression construct or vector as a control along with β-galactosidase expression plasmid. Cells were treated with TNF (20 ng/ml) for 6 h before harvesting them for luciferase activity. Relative Luc activity, normalized to the corresponding β-galactosidase activity, is shown (B).

    Journal: Molecular and Cellular Biology

    Article Title: Suppression of PTEN Expression by NF-?B Prevents Apoptosis

    doi: 10.1128/MCB.24.3.1007-1021.2004

    Figure Lengend Snippet: NF-κB down-regulates the PTEN promoter. NIH 3T3 cells were cotransfected with pGL3 basic-Luc, PTEN promoter-Luc, or cyclin D1-Luc reporter construct, as indicated, and p65 expression construct or vector as a control along with β-galactosidase expression plasmid. Relative Luc activity, normalized to the corresponding β-galactosidase activity is shown (A). Relative Luc activity of 100 corresponds to 16,000 to 25,000 U of the raw Luc activity values normalized to β-galactosidase activity for all of the luciferase reporter experiments. NIH 3T3 cells were cotransfected with the PTEN promoter-Luc reporter construct or NF-κB-Luc reporter construct, as indicated, and IκB-SR expression construct or vector as a control along with β-galactosidase expression plasmid. Cells were treated with TNF (20 ng/ml) for 6 h before harvesting them for luciferase activity. Relative Luc activity, normalized to the corresponding β-galactosidase activity, is shown (B).

    Article Snippet: Mouse monoclonal antibodies for PTEN (A2B1) and IKKβ (H-4) and rabbit polyclonal antibodies for p65 (C-20), p50 (H-119), IκB-α (C-21), Akt1/2 (H-136), X chromosome-linked IAP (XIAP) (H-202), and cIAP1 (H-83) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.).

    Techniques: Construct, Expressing, Plasmid Preparation, Activity Assay, Luciferase

    PTEN inhibits transactivation and antiapoptotic functions of p65. NIH 3T3 cells were cotransfected with Gal4-p65 fusion construct as a driver and 5× Gal4-Luc reporter in the presence or absence of vector, wild-type PTEN, or the phosphatase-defective C124S mutant of PTEN. Luciferase activity was determined at 48 h posttransfection (A). Relative Luc activity, normalized to the corresponding β-galactosidase activity, is shown. Cell extracts from PTEN +/+ and PTEN −/− MEFs were subjected to Western blot analysis for expression of XIAP, PTEN, and actin (B). NIH 3T3 cells were transfected with GFP-vector, GFP-PTEN, myr-Akt1, or DN-Akt1, and 24 h after transfection, the cultures were treated with either vehicle or murine TNF (20 ng/ml) and incubated for another 24 h. Apoptotic cells were scored as described in Materials and Methods (C). Whole-cell extracts prepared from transfected cells where subjected to Western blot analysis for expression of Akt, PTEN, and actin (C). A model for the negative regulatory loop between NF-κB and PTEN involved in the modulation of TNF-inducible apoptosis is depicted (D). TNF-inducible apoptosis is known to be prevented by NF-κB activation. We suggest that activation of NF-κB by TNF results in down-regulation of PTEN expression and prevention of apoptosis. Overexpression of PTEN results in inhibition of NF-κB transcriptional activity and potentiation of TNF-inducible apoptosis (D).

    Journal: Molecular and Cellular Biology

    Article Title: Suppression of PTEN Expression by NF-?B Prevents Apoptosis

    doi: 10.1128/MCB.24.3.1007-1021.2004

    Figure Lengend Snippet: PTEN inhibits transactivation and antiapoptotic functions of p65. NIH 3T3 cells were cotransfected with Gal4-p65 fusion construct as a driver and 5× Gal4-Luc reporter in the presence or absence of vector, wild-type PTEN, or the phosphatase-defective C124S mutant of PTEN. Luciferase activity was determined at 48 h posttransfection (A). Relative Luc activity, normalized to the corresponding β-galactosidase activity, is shown. Cell extracts from PTEN +/+ and PTEN −/− MEFs were subjected to Western blot analysis for expression of XIAP, PTEN, and actin (B). NIH 3T3 cells were transfected with GFP-vector, GFP-PTEN, myr-Akt1, or DN-Akt1, and 24 h after transfection, the cultures were treated with either vehicle or murine TNF (20 ng/ml) and incubated for another 24 h. Apoptotic cells were scored as described in Materials and Methods (C). Whole-cell extracts prepared from transfected cells where subjected to Western blot analysis for expression of Akt, PTEN, and actin (C). A model for the negative regulatory loop between NF-κB and PTEN involved in the modulation of TNF-inducible apoptosis is depicted (D). TNF-inducible apoptosis is known to be prevented by NF-κB activation. We suggest that activation of NF-κB by TNF results in down-regulation of PTEN expression and prevention of apoptosis. Overexpression of PTEN results in inhibition of NF-κB transcriptional activity and potentiation of TNF-inducible apoptosis (D).

    Article Snippet: Mouse monoclonal antibodies for PTEN (A2B1) and IKKβ (H-4) and rabbit polyclonal antibodies for p65 (C-20), p50 (H-119), IκB-α (C-21), Akt1/2 (H-136), X chromosome-linked IAP (XIAP) (H-202), and cIAP1 (H-83) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.).

    Techniques: Construct, Plasmid Preparation, Mutagenesis, Luciferase, Activity Assay, Western Blot, Expressing, Transfection, Incubation, Activation Assay, Over Expression, Inhibition

    PTEN is down-regulated by NF-κB. p65 −/− immortalized MEFs were transiently transfected for 48 h with constructs expressing p50, p65, or vector (as a control), and the expression of p65, PTEN, or actin (as loading control) was examined by Western blot analysis (A). The signal intensity was quantified by densitometry and normalized to the corresponding signal for actin, and the relative PTEN protein levels (in arbitrary units) are shown at the bottom of panel A. p65 −/− cells were transiently transfected for 48 h with constructs expressing p50, p65, or vector (as a control), and expression of phospho-Akt, total Akt, p65, p50, or actin was examined by Western blot analysis (B). NIH 3T3 cells were treated with murine TNF or IL-1, and whole-cell protein extracts prepared after 24 h were subjected to Western blot analysis for PTEN, cIAP1, or actin (C). Human MCF-7 breast cancer cells were treated with human TNF, and total RNA, prepared after the indicated time points, was subjected to Northern blot analysis for PTEN or GAPDH (as a control) (D). The RNA signal intensity was quantified by densitometry and normalized to the corresponding signal for GAPDH, and the relative PTEN RNA levels (in arbitrary units) are shown at the bottom of panel D. MCF-7 cells were treated with human TNF for the indicated time points, and whole-cell protein extracts were subjected to Western blot analysis for PTEN and actin (D). NIH 3T3 cells were treated with murine TNF for the indicated time periods, and total RNA or whole-cell lysates were subjected to either Northern blot analysis for PTEN (E) or Western blot analysis for PTEN and actin expression (E). The RNA signal intensity was quantified by densitometry and normalized to the corresponding signal for 28S RNA on the corresponding ethidium bromide gel, and the relative PTEN RNA levels (in arbitrary units) are shown at the bottom of panel E. IKKβ −/− or wild-type (IKKβ +/+ ) MEFs were treated with murine TNF or vehicle for 24 h, and whole-cell extracts were analyzed for PTEN, IKKβ, or actin expression by Western blot analysis (F). NIH 3T3 cells were transfected with GFP-PTEN expression plasmid along with vector or p65 expression construct used at a 1:1 ratio, and whole-cell protein extracts prepared after 36 h were subjected to Western blot analysis for expression of PTEN or actin (G).

    Journal: Molecular and Cellular Biology

    Article Title: Suppression of PTEN Expression by NF-?B Prevents Apoptosis

    doi: 10.1128/MCB.24.3.1007-1021.2004

    Figure Lengend Snippet: PTEN is down-regulated by NF-κB. p65 −/− immortalized MEFs were transiently transfected for 48 h with constructs expressing p50, p65, or vector (as a control), and the expression of p65, PTEN, or actin (as loading control) was examined by Western blot analysis (A). The signal intensity was quantified by densitometry and normalized to the corresponding signal for actin, and the relative PTEN protein levels (in arbitrary units) are shown at the bottom of panel A. p65 −/− cells were transiently transfected for 48 h with constructs expressing p50, p65, or vector (as a control), and expression of phospho-Akt, total Akt, p65, p50, or actin was examined by Western blot analysis (B). NIH 3T3 cells were treated with murine TNF or IL-1, and whole-cell protein extracts prepared after 24 h were subjected to Western blot analysis for PTEN, cIAP1, or actin (C). Human MCF-7 breast cancer cells were treated with human TNF, and total RNA, prepared after the indicated time points, was subjected to Northern blot analysis for PTEN or GAPDH (as a control) (D). The RNA signal intensity was quantified by densitometry and normalized to the corresponding signal for GAPDH, and the relative PTEN RNA levels (in arbitrary units) are shown at the bottom of panel D. MCF-7 cells were treated with human TNF for the indicated time points, and whole-cell protein extracts were subjected to Western blot analysis for PTEN and actin (D). NIH 3T3 cells were treated with murine TNF for the indicated time periods, and total RNA or whole-cell lysates were subjected to either Northern blot analysis for PTEN (E) or Western blot analysis for PTEN and actin expression (E). The RNA signal intensity was quantified by densitometry and normalized to the corresponding signal for 28S RNA on the corresponding ethidium bromide gel, and the relative PTEN RNA levels (in arbitrary units) are shown at the bottom of panel E. IKKβ −/− or wild-type (IKKβ +/+ ) MEFs were treated with murine TNF or vehicle for 24 h, and whole-cell extracts were analyzed for PTEN, IKKβ, or actin expression by Western blot analysis (F). NIH 3T3 cells were transfected with GFP-PTEN expression plasmid along with vector or p65 expression construct used at a 1:1 ratio, and whole-cell protein extracts prepared after 36 h were subjected to Western blot analysis for expression of PTEN or actin (G).

    Article Snippet: Mouse monoclonal antibodies for PTEN (A2B1) and IKKβ (H-4) and rabbit polyclonal antibodies for p65 (C-20), p50 (H-119), IκB-α (C-21), Akt1/2 (H-136), X chromosome-linked IAP (XIAP) (H-202), and cIAP1 (H-83) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.).

    Techniques: Transfection, Construct, Expressing, Plasmid Preparation, Western Blot, Northern Blot

    Elevated levels of p65 protein in certain human lung and thyroid cancer cells repress PTEN expression. Whole-cell extracts were prepared from non-small-cell lung carcinoma or thyroid cancer cell lines and subjected to Western blot analysis for p65, PTEN, or actin (A). MRO and A549 cells were cotransfected with the PTEN promoter-Luc reporter construct and IκB-SR expression constructs or vector as a control along with β-galactosidase expression plasmid. Relative Luc activity measured after 48 h of transfection, normalized to the corresponding β-galactosidase activity, is shown (B). A549 and H838 lung cancer cells and MRO and ARO thyroid cancer cells were infected with adenovirus expression vector for IκB-SR or GFP as a control, and whole-cell protein extracts collected after 24 or 48 h were subjected to Western blot analysis for PTEN, IκB-α, or actin. Cell extracts from PTEN +/+ and PTEN −/− MEFs were loaded as controls for PTEN expression (C).

    Journal: Molecular and Cellular Biology

    Article Title: Suppression of PTEN Expression by NF-?B Prevents Apoptosis

    doi: 10.1128/MCB.24.3.1007-1021.2004

    Figure Lengend Snippet: Elevated levels of p65 protein in certain human lung and thyroid cancer cells repress PTEN expression. Whole-cell extracts were prepared from non-small-cell lung carcinoma or thyroid cancer cell lines and subjected to Western blot analysis for p65, PTEN, or actin (A). MRO and A549 cells were cotransfected with the PTEN promoter-Luc reporter construct and IκB-SR expression constructs or vector as a control along with β-galactosidase expression plasmid. Relative Luc activity measured after 48 h of transfection, normalized to the corresponding β-galactosidase activity, is shown (B). A549 and H838 lung cancer cells and MRO and ARO thyroid cancer cells were infected with adenovirus expression vector for IκB-SR or GFP as a control, and whole-cell protein extracts collected after 24 or 48 h were subjected to Western blot analysis for PTEN, IκB-α, or actin. Cell extracts from PTEN +/+ and PTEN −/− MEFs were loaded as controls for PTEN expression (C).

    Article Snippet: Mouse monoclonal antibodies for PTEN (A2B1) and IKKβ (H-4) and rabbit polyclonal antibodies for p65 (C-20), p50 (H-119), IκB-α (C-21), Akt1/2 (H-136), X chromosome-linked IAP (XIAP) (H-202), and cIAP1 (H-83) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.).

    Techniques: Expressing, Western Blot, Construct, Plasmid Preparation, Activity Assay, Transfection, Infection

    Putative NF-κB binding sites in the PTEN promoter do not mediate repression by p65. NIH 3T3 cells were cotransfected with either full-length PTEN promoter-Luc reporter construct or Δ600 PTEN promoter-Luc reporter construct, in which the two putative NF-κB binding sites were deleted, along with either vector or p65 constructs, and luciferase activity was determined at 48 h posttransfection (A). NIH 3T3 cells were cotransfected with either empty Tal-Luc reporter construct or PTEN 600 Tal-Luc reporter construct, which contained the 600-bp region of the PTEN promoter with the two putative NF-κB binding sites subcloned in Tal-Luc, along with either vector or p65 constructs, and luciferase activity was determined at 48 h posttransfection (B). Relative Luc activity, normalized to the corresponding β-galactosidase activity, is shown (A and B).

    Journal: Molecular and Cellular Biology

    Article Title: Suppression of PTEN Expression by NF-?B Prevents Apoptosis

    doi: 10.1128/MCB.24.3.1007-1021.2004

    Figure Lengend Snippet: Putative NF-κB binding sites in the PTEN promoter do not mediate repression by p65. NIH 3T3 cells were cotransfected with either full-length PTEN promoter-Luc reporter construct or Δ600 PTEN promoter-Luc reporter construct, in which the two putative NF-κB binding sites were deleted, along with either vector or p65 constructs, and luciferase activity was determined at 48 h posttransfection (A). NIH 3T3 cells were cotransfected with either empty Tal-Luc reporter construct or PTEN 600 Tal-Luc reporter construct, which contained the 600-bp region of the PTEN promoter with the two putative NF-κB binding sites subcloned in Tal-Luc, along with either vector or p65 constructs, and luciferase activity was determined at 48 h posttransfection (B). Relative Luc activity, normalized to the corresponding β-galactosidase activity, is shown (A and B).

    Article Snippet: Mouse monoclonal antibodies for PTEN (A2B1) and IKKβ (H-4) and rabbit polyclonal antibodies for p65 (C-20), p50 (H-119), IκB-α (C-21), Akt1/2 (H-136), X chromosome-linked IAP (XIAP) (H-202), and cIAP1 (H-83) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.).

    Techniques: Binding Assay, Construct, Plasmid Preparation, Luciferase, Activity Assay

    Cotransfection of CBP or p300 relieves p65-mediated repression of PTEN. NIH 3T3 cells were cotransfected with the PTEN promoter-Luc reporter construct and p65 expression construct alone or vector for control or with a combination of p65 and wild-type p300 or CBP expression constructs (A, left panel) or a combination of p65 and p300-C, an amino-terminal deletion mutant of p300 lacking the C/H1 and KIX domains (A, upper panel), used at a 1:1 ratio, along with β-galactosidase expression plasmid. Relative Luc activity, normalized to the corresponding β-galactosidase activity, is shown (A). A model for the mechanism of PTEN suppression by NF-κB is shown (B). In unstimulated cells, the NF-κB complex consisting of p65 and p50 is sequestered by IκB. Stimulation with proinflammatory cytokines such as TNF or IL-1 causes phosphorylation and degradation of IκB, with the release of p65/p50 complex resulting in exposure of the serine-276 residue on p65. Phosphorylation of p65 at the serine-276 site by PKA results in efficient binding with, and sequestration of, transcriptional coactivators CBP/p300, thereby blocking the transcriptional activation by certain transcription factors responsible for basal and PKA-inducible expression of the PTEN gene and causing repression of PTEN. It is currently unknown which one of the PKA-activated transcription factors are targeted by p65 via CBP or p300 sequestration. Transcription factors C/EBP-α and -β and RXR are potential targets with 3 sites for C/EBP (at nucleotide positions −1311, −1208, and −819; shown in pink), and 4 sites for RXR (at nucleotide positions −1377, −1227, −1028, and −977; shown in purple) present in the PTEN promoter region. The transcription start site is at position −1031.

    Journal: Molecular and Cellular Biology

    Article Title: Suppression of PTEN Expression by NF-?B Prevents Apoptosis

    doi: 10.1128/MCB.24.3.1007-1021.2004

    Figure Lengend Snippet: Cotransfection of CBP or p300 relieves p65-mediated repression of PTEN. NIH 3T3 cells were cotransfected with the PTEN promoter-Luc reporter construct and p65 expression construct alone or vector for control or with a combination of p65 and wild-type p300 or CBP expression constructs (A, left panel) or a combination of p65 and p300-C, an amino-terminal deletion mutant of p300 lacking the C/H1 and KIX domains (A, upper panel), used at a 1:1 ratio, along with β-galactosidase expression plasmid. Relative Luc activity, normalized to the corresponding β-galactosidase activity, is shown (A). A model for the mechanism of PTEN suppression by NF-κB is shown (B). In unstimulated cells, the NF-κB complex consisting of p65 and p50 is sequestered by IκB. Stimulation with proinflammatory cytokines such as TNF or IL-1 causes phosphorylation and degradation of IκB, with the release of p65/p50 complex resulting in exposure of the serine-276 residue on p65. Phosphorylation of p65 at the serine-276 site by PKA results in efficient binding with, and sequestration of, transcriptional coactivators CBP/p300, thereby blocking the transcriptional activation by certain transcription factors responsible for basal and PKA-inducible expression of the PTEN gene and causing repression of PTEN. It is currently unknown which one of the PKA-activated transcription factors are targeted by p65 via CBP or p300 sequestration. Transcription factors C/EBP-α and -β and RXR are potential targets with 3 sites for C/EBP (at nucleotide positions −1311, −1208, and −819; shown in pink), and 4 sites for RXR (at nucleotide positions −1377, −1227, −1028, and −977; shown in purple) present in the PTEN promoter region. The transcription start site is at position −1031.

    Article Snippet: Mouse monoclonal antibodies for PTEN (A2B1) and IKKβ (H-4) and rabbit polyclonal antibodies for p65 (C-20), p50 (H-119), IκB-α (C-21), Akt1/2 (H-136), X chromosome-linked IAP (XIAP) (H-202), and cIAP1 (H-83) were from Santa Cruz Biotechnology, Inc. (Santa Cruz, Calif.).

    Techniques: Cotransfection, Construct, Expressing, Plasmid Preparation, Mutagenesis, Activity Assay, Binding Assay, Blocking Assay, Activation Assay

    siRNA-mediated knock-down of MSK1 in H292 cells attenuated the phosphorylation of RelA/p65 by CSE treatment. MSK1 knock-down by siRNA approach attenuated the phosphorylation of RelA/p65 by CSE in human bronchial epithelial cells. H292 cells were transfected with 100 nM scrambled siRNA (non-targeted siRNA) or siRNA directed against MSK1 (MSK1 siRNA) for 24 h, followed by starvation in serum free medium and treated for 1 h after 72 h of transfection with CSE (1%). Whole cell lysate was prepared after 1 h treatment and assayed for effect of MSK1 knock-down by immunoblotting. The levels of p-MSK1 (Thr581), total MSK1, phosphorylated RelA/p65 (Ser276), acetylated RelA/p65 (Lys310), and total RelA/p65 were determined by immunoblotting. β-actin was used as a loading control. Gel pictures are representative of at least three separate experiments. The band intensity was measured by densitometry and data shown as fold change relative to β-actin control. Data are shown as mean ± SEM; *, P

    Journal: PLoS ONE

    Article Title: Mitogen- and Stress-Activated Kinase 1 (MSK1) Regulates Cigarette Smoke-Induced Histone Modifications on NF-?B-dependent Genes

    doi: 10.1371/journal.pone.0031378

    Figure Lengend Snippet: siRNA-mediated knock-down of MSK1 in H292 cells attenuated the phosphorylation of RelA/p65 by CSE treatment. MSK1 knock-down by siRNA approach attenuated the phosphorylation of RelA/p65 by CSE in human bronchial epithelial cells. H292 cells were transfected with 100 nM scrambled siRNA (non-targeted siRNA) or siRNA directed against MSK1 (MSK1 siRNA) for 24 h, followed by starvation in serum free medium and treated for 1 h after 72 h of transfection with CSE (1%). Whole cell lysate was prepared after 1 h treatment and assayed for effect of MSK1 knock-down by immunoblotting. The levels of p-MSK1 (Thr581), total MSK1, phosphorylated RelA/p65 (Ser276), acetylated RelA/p65 (Lys310), and total RelA/p65 were determined by immunoblotting. β-actin was used as a loading control. Gel pictures are representative of at least three separate experiments. The band intensity was measured by densitometry and data shown as fold change relative to β-actin control. Data are shown as mean ± SEM; *, P

    Article Snippet: MSK1, p-S276 (RelA/p65), RelA/p65, and Lamin B antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Transfection

    MSK1 interacted with RelA/p65 and p300. (A) H292 cells were transiently transfected with wild-type MSK1 or MSK1 N-terminal kinase-dead mutant MSK1 flag construct and treated with or without CSE (1.0%) for 1 h. Flag-tagged precipitated complexes were analyzed through immunoblotting using anti-flag and anti-RelA/p65 antibodies. Whole cell lysate from pCMV-Flag-MSK1 wild-type transfected cells were used as lysate control. (B) H292 cells were treated for 1 h with or without CSE (2.0%) and TNFα (10 ng/ml) was used as positive control. Nuclear extract (1000 µg) was used for immunoprecipitation of MSK1 bound complexes and analyzed through immunoblotting using anti-MSK1, anti-RelA/p65 and anti-p300 antibodies. (C) H292 cells were treated for 1 h with or without CSE (2.0%), and TNFα (10 ng/ml) was used as positive control. Nuclear extract (1000 µg) was used for immunoprecipitation of RelA/p65 bound complexes and analyzed through immunoblotting using anti-RelA/p65 and anti-MSK1 antibodies. (D) CS-mediated interaction of MSK1-p300 in acute CS exposed mouse lung. Adult C57BL/6J mice were exposed to CS at 300 mg/m 3 total particulate matter (TPM) for 3 days and were sacrificed 24 h after the final exposure. Whole tissue extract (500 µg) was used for immunoprecipitation of MSK1 bound complexes, and analyzed by immunoblotting using anti-MSK1 and anti-p300 antibodies. (E) CS-mediated interaction of RelA/p65 with MSK1 and p300 in acute CS exposed mouse lung. Adult C57BL/6J mice were exposed to CS at 300 mg/m 3 total particulate matter (TPM) for 3 days and were sacrificed 24 h after the final exposure. Whole tissue extract (500 µg) was used for immunoprecipitation of RelA/p65 bound complexes, and analyzed by immunoblotting using anti-RelA/p65, anti-MSK1 and anti-p300 antibodies. Results are representative of at least three separate experiments.

    Journal: PLoS ONE

    Article Title: Mitogen- and Stress-Activated Kinase 1 (MSK1) Regulates Cigarette Smoke-Induced Histone Modifications on NF-?B-dependent Genes

    doi: 10.1371/journal.pone.0031378

    Figure Lengend Snippet: MSK1 interacted with RelA/p65 and p300. (A) H292 cells were transiently transfected with wild-type MSK1 or MSK1 N-terminal kinase-dead mutant MSK1 flag construct and treated with or without CSE (1.0%) for 1 h. Flag-tagged precipitated complexes were analyzed through immunoblotting using anti-flag and anti-RelA/p65 antibodies. Whole cell lysate from pCMV-Flag-MSK1 wild-type transfected cells were used as lysate control. (B) H292 cells were treated for 1 h with or without CSE (2.0%) and TNFα (10 ng/ml) was used as positive control. Nuclear extract (1000 µg) was used for immunoprecipitation of MSK1 bound complexes and analyzed through immunoblotting using anti-MSK1, anti-RelA/p65 and anti-p300 antibodies. (C) H292 cells were treated for 1 h with or without CSE (2.0%), and TNFα (10 ng/ml) was used as positive control. Nuclear extract (1000 µg) was used for immunoprecipitation of RelA/p65 bound complexes and analyzed through immunoblotting using anti-RelA/p65 and anti-MSK1 antibodies. (D) CS-mediated interaction of MSK1-p300 in acute CS exposed mouse lung. Adult C57BL/6J mice were exposed to CS at 300 mg/m 3 total particulate matter (TPM) for 3 days and were sacrificed 24 h after the final exposure. Whole tissue extract (500 µg) was used for immunoprecipitation of MSK1 bound complexes, and analyzed by immunoblotting using anti-MSK1 and anti-p300 antibodies. (E) CS-mediated interaction of RelA/p65 with MSK1 and p300 in acute CS exposed mouse lung. Adult C57BL/6J mice were exposed to CS at 300 mg/m 3 total particulate matter (TPM) for 3 days and were sacrificed 24 h after the final exposure. Whole tissue extract (500 µg) was used for immunoprecipitation of RelA/p65 bound complexes, and analyzed by immunoblotting using anti-RelA/p65, anti-MSK1 and anti-p300 antibodies. Results are representative of at least three separate experiments.

    Article Snippet: MSK1, p-S276 (RelA/p65), RelA/p65, and Lamin B antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Transfection, Mutagenesis, Construct, Positive Control, Immunoprecipitation, Mouse Assay

    A schematic model showing the role of MSK1 in cigarette smoke-induced NF-κB signaling and chromatin modifications. Cigarette smoke/aldehyde/oxidative stress via activation of MSK1 results in phosphorylation, and nuclear transclocation of NF-κB RelA/p65. Activated nuclear MSK1 subsequently causes chromatin modifications of histones (phospho-acetylation of histone H3 [Ser10/Lys9] and acetylation of histone H4 [Lys12]). MSK1 N-terminal kinase dead mutant, siRNA-mediated knock-down and stable MSK1 knock-down MEFs represses the nuclear MSK1, phosphorylation of RelA/p65, and phospho-acetylation of histone H3 and acetylation of histone H4. CS activates MSK1 which forms a complex with NF-κB RelA/p65 and coactivator p300 which further mediates transcriptional activation of NF-κB-dependent pro-inflammatory genes (IL-6, IL-8 and COX-2). (↑, Induction; ⊢ inhibition).

    Journal: PLoS ONE

    Article Title: Mitogen- and Stress-Activated Kinase 1 (MSK1) Regulates Cigarette Smoke-Induced Histone Modifications on NF-?B-dependent Genes

    doi: 10.1371/journal.pone.0031378

    Figure Lengend Snippet: A schematic model showing the role of MSK1 in cigarette smoke-induced NF-κB signaling and chromatin modifications. Cigarette smoke/aldehyde/oxidative stress via activation of MSK1 results in phosphorylation, and nuclear transclocation of NF-κB RelA/p65. Activated nuclear MSK1 subsequently causes chromatin modifications of histones (phospho-acetylation of histone H3 [Ser10/Lys9] and acetylation of histone H4 [Lys12]). MSK1 N-terminal kinase dead mutant, siRNA-mediated knock-down and stable MSK1 knock-down MEFs represses the nuclear MSK1, phosphorylation of RelA/p65, and phospho-acetylation of histone H3 and acetylation of histone H4. CS activates MSK1 which forms a complex with NF-κB RelA/p65 and coactivator p300 which further mediates transcriptional activation of NF-κB-dependent pro-inflammatory genes (IL-6, IL-8 and COX-2). (↑, Induction; ⊢ inhibition).

    Article Snippet: MSK1, p-S276 (RelA/p65), RelA/p65, and Lamin B antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Activation Assay, Mutagenesis, Inhibition

    MSK1 N- and C-terminal kinase-dead mutants in H292 cells attenuated the phosphorylation of RelA/p65 by CSE treatment. Cigarette smoke increased the cellular levels of phosphorylated MSK1 as well as phosphorylated RelA/p65 levels in control vector (pCMV-FLAG) and MSK1 WT (pCMV-FLAG-MSK1 wild-type) transfected human bronchial epithelial cells compared to MSK1 N- and C-terminal kinase-dead mutants (pCMV-FLAG-MSK1 ND and pCMV-FLAG-MSK1 CD). H292 cells were transiently transfected with control vector, MSK1 WT, MSK1 N-terminal kinase-dead (ND) mutant, MSK1 C-terminal kinase-dead (CD) mutant and treated with or without CSE (1%) after 24-48 hrs of transfection for 1 h. Cells were harvested (1 h later). Whole cell extracts were prepared and immunoblotted for p-MSK1 (Thr581), total MSK1, phosphorylated RelA/p65 (Ser276), acetylated RelA/p65 (Lys310), and total RelA/p65. β-actin was used as protein loading control. Gel pictures are representative of at least three separate experiments. The band intensity was measured by densitometry and data shown as fold change relative to β-actin control. Data are shown as mean ± SEM; **, P

    Journal: PLoS ONE

    Article Title: Mitogen- and Stress-Activated Kinase 1 (MSK1) Regulates Cigarette Smoke-Induced Histone Modifications on NF-?B-dependent Genes

    doi: 10.1371/journal.pone.0031378

    Figure Lengend Snippet: MSK1 N- and C-terminal kinase-dead mutants in H292 cells attenuated the phosphorylation of RelA/p65 by CSE treatment. Cigarette smoke increased the cellular levels of phosphorylated MSK1 as well as phosphorylated RelA/p65 levels in control vector (pCMV-FLAG) and MSK1 WT (pCMV-FLAG-MSK1 wild-type) transfected human bronchial epithelial cells compared to MSK1 N- and C-terminal kinase-dead mutants (pCMV-FLAG-MSK1 ND and pCMV-FLAG-MSK1 CD). H292 cells were transiently transfected with control vector, MSK1 WT, MSK1 N-terminal kinase-dead (ND) mutant, MSK1 C-terminal kinase-dead (CD) mutant and treated with or without CSE (1%) after 24-48 hrs of transfection for 1 h. Cells were harvested (1 h later). Whole cell extracts were prepared and immunoblotted for p-MSK1 (Thr581), total MSK1, phosphorylated RelA/p65 (Ser276), acetylated RelA/p65 (Lys310), and total RelA/p65. β-actin was used as protein loading control. Gel pictures are representative of at least three separate experiments. The band intensity was measured by densitometry and data shown as fold change relative to β-actin control. Data are shown as mean ± SEM; **, P

    Article Snippet: MSK1, p-S276 (RelA/p65), RelA/p65, and Lamin B antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Plasmid Preparation, Transfection, Mutagenesis

    ChIP assay revealed that MSK1 is localized to pro-inflammatory gene promoters (IL-8, IL-6 and COX-2). H292 cells were treated with CSE (1%) for 1 h. ChIP analysis was performed against MSK1, phospho-RelA/p65 (Ser276), phospho-histone H3 (Lys9) and acetylated histone H4 (Lys12) antibodies. After reverse cross-linking, co-immunoprecipitated genomic DNA fragments were analyzed by semi-quantitative PCR with IL-6, IL-8, and COX-2 promoter specific primer sets. IgG was used as negative control, and RNA Polymerase II antibody was used as positive control. Input reflects the relative amount of sonicated DNA fragments present before immunoprecipitation and revealed by semi-quantitative PCR with pro-inflammatory gene specific primers. The band intensity was measured by densitometry and data shown as fold change versus input. Data are shown as mean ± SEM (n = 3). *, P

    Journal: PLoS ONE

    Article Title: Mitogen- and Stress-Activated Kinase 1 (MSK1) Regulates Cigarette Smoke-Induced Histone Modifications on NF-?B-dependent Genes

    doi: 10.1371/journal.pone.0031378

    Figure Lengend Snippet: ChIP assay revealed that MSK1 is localized to pro-inflammatory gene promoters (IL-8, IL-6 and COX-2). H292 cells were treated with CSE (1%) for 1 h. ChIP analysis was performed against MSK1, phospho-RelA/p65 (Ser276), phospho-histone H3 (Lys9) and acetylated histone H4 (Lys12) antibodies. After reverse cross-linking, co-immunoprecipitated genomic DNA fragments were analyzed by semi-quantitative PCR with IL-6, IL-8, and COX-2 promoter specific primer sets. IgG was used as negative control, and RNA Polymerase II antibody was used as positive control. Input reflects the relative amount of sonicated DNA fragments present before immunoprecipitation and revealed by semi-quantitative PCR with pro-inflammatory gene specific primers. The band intensity was measured by densitometry and data shown as fold change versus input. Data are shown as mean ± SEM (n = 3). *, P

    Article Snippet: MSK1, p-S276 (RelA/p65), RelA/p65, and Lamin B antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Chromatin Immunoprecipitation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Negative Control, Positive Control, Sonication

    Cigarette smoke increased the nuclear levels of phosphorylated MSK1, phosphorylated and acetylated RelA/p65 in a time-dependent manner in human bronchial and small airway epithelial cells, and in mouse lung. (A) H292, BEAS-2B and SAEC cells were treated with or without 1% CSE (time course: 0, 15, 30, and 60 minutes). Cells were harvested, nuclear extracts were prepared and immunoblotted for p-MSK1 (Thr581), total MSK1, phosphorylated RelA/p65 (Ser276), acetylated RelA/p65 (Lys310), and total RelA/p65. Lamin B was used as nuclear protein loading controls. Gel pictures shown are representative of at least three separate experiments. (B) The band intensity was measured by densitometry and data shown as fold change relative to Lamin B control. Data are shown as mean ± SEM; *, P

    Journal: PLoS ONE

    Article Title: Mitogen- and Stress-Activated Kinase 1 (MSK1) Regulates Cigarette Smoke-Induced Histone Modifications on NF-?B-dependent Genes

    doi: 10.1371/journal.pone.0031378

    Figure Lengend Snippet: Cigarette smoke increased the nuclear levels of phosphorylated MSK1, phosphorylated and acetylated RelA/p65 in a time-dependent manner in human bronchial and small airway epithelial cells, and in mouse lung. (A) H292, BEAS-2B and SAEC cells were treated with or without 1% CSE (time course: 0, 15, 30, and 60 minutes). Cells were harvested, nuclear extracts were prepared and immunoblotted for p-MSK1 (Thr581), total MSK1, phosphorylated RelA/p65 (Ser276), acetylated RelA/p65 (Lys310), and total RelA/p65. Lamin B was used as nuclear protein loading controls. Gel pictures shown are representative of at least three separate experiments. (B) The band intensity was measured by densitometry and data shown as fold change relative to Lamin B control. Data are shown as mean ± SEM; *, P

    Article Snippet: MSK1, p-S276 (RelA/p65), RelA/p65, and Lamin B antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques:

    MSK1 knock-down by MSK1 siRNA in H292 cells attenuated the phosphorylation of RelA/p65 by CSE treatment demonstrated by immunocytochemistry. CSE-induced activation of MSK1 and RelA/p65 (Ser276) in non-targeted siRNA transfected human bronchial epithelial cells. MSK1 knock-down by siRNA approach attenuated the activation of MSK1 and phosphorylation of RelA/p65 by CSE in human bronchial epithelial cells. H292 cells were transfected with 100 nM scrambled siRNA (non-targeted siRNA) or MSK1 siRNA for 24 h, followed by starvation in serum free medium and then treated for 1 h (72 h post-transfection) with CSE (1.0%). For immunocytochemistry, cells were fixed, permeabilized, and stained for the expression of p-MSK1 (Thr581), total MSK1, and phosphorylated RelA/p65 (Ser276) by immunofluorescence. Phosphorylated MSK1, total MSK1, and phosphorylated RelA/p65 are shown in red, and DNA (Hoechst nuclear staining) in blue, and merge represented as dark purple or pink. Results are representative cells from at least three separate experiments. The quantification of fluorescence intensity in immunofluorescence data was measured using ImageJ and the corrected total cell fluorescence (CTCF) values were converted into fold change values and represented as histograms. Data are shown as mean ± SEM; **, P

    Journal: PLoS ONE

    Article Title: Mitogen- and Stress-Activated Kinase 1 (MSK1) Regulates Cigarette Smoke-Induced Histone Modifications on NF-?B-dependent Genes

    doi: 10.1371/journal.pone.0031378

    Figure Lengend Snippet: MSK1 knock-down by MSK1 siRNA in H292 cells attenuated the phosphorylation of RelA/p65 by CSE treatment demonstrated by immunocytochemistry. CSE-induced activation of MSK1 and RelA/p65 (Ser276) in non-targeted siRNA transfected human bronchial epithelial cells. MSK1 knock-down by siRNA approach attenuated the activation of MSK1 and phosphorylation of RelA/p65 by CSE in human bronchial epithelial cells. H292 cells were transfected with 100 nM scrambled siRNA (non-targeted siRNA) or MSK1 siRNA for 24 h, followed by starvation in serum free medium and then treated for 1 h (72 h post-transfection) with CSE (1.0%). For immunocytochemistry, cells were fixed, permeabilized, and stained for the expression of p-MSK1 (Thr581), total MSK1, and phosphorylated RelA/p65 (Ser276) by immunofluorescence. Phosphorylated MSK1, total MSK1, and phosphorylated RelA/p65 are shown in red, and DNA (Hoechst nuclear staining) in blue, and merge represented as dark purple or pink. Results are representative cells from at least three separate experiments. The quantification of fluorescence intensity in immunofluorescence data was measured using ImageJ and the corrected total cell fluorescence (CTCF) values were converted into fold change values and represented as histograms. Data are shown as mean ± SEM; **, P

    Article Snippet: MSK1, p-S276 (RelA/p65), RelA/p65, and Lamin B antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Immunocytochemistry, Activation Assay, Transfection, Staining, Expressing, Immunofluorescence, Fluorescence

    Cigarette smoke increased p-MSK1 (Thr581) and p-RelA/p65 (Ser276) in human bronchial and small airway epithelial cells as demonstrated by immunocytochemistry, and in mouse lung by immunoblotting. (A) Activation of MSK1 and RelA/p65 (Ser276) in H292 cells treated with CSE (1%) for 1 h. For immunocytochemistry, cells were fixed, permeabilized, and stained for the expression of p-MSK1 (Thr581), total MSK1, and phosphorylated RelA/p65 (Ser276) by immunofluorescence. Phosphorylated MSK1, total MSK1, and phosphorylated RelA/p65 is shown in red, and DNA (Hoechst nuclear staining) in blue, and merge represented as dark purple or pink. Results are representative cells from three separate experiments. The panel without primary antibodies was considered as negative control (NC). (B) Activation of MSK1 and RelA/p65 (Ser276) in SAEC cells treated with CSE (1%) for 1 h. For immunocytochemistry, cells were fixed, permeabilized, and stained for the expression of p-MSK1 (Thr581), total MSK1, and phosphorylated RelA/p65 (Ser276) by immunofluorescence. Phosphorylated MSK1, and phosphorylated RelA/p65 is shown in red, total MSK1 in green, and DNA (Hoechst nuclear staining) in blue, and merge represented as dark purple or pink. Results are representative cells from three separate experiments. The panel without primary antibodies was considered as negative control (NC). (C) Nuclear levels of MSK1 (phosphorylated and total MSK1) were increased in lung tissue of mouse exposed to CS determined by immunoblot analysis. Adult C57BL/6J mice were exposed to CS for 3 days and were sacrificed 24 hrs post-final CS exposure. Lamin B was used as protein loading control. The band intensity was measured by densitometry and data shown as fold change relative to Lamin B control. Data are shown as mean ± SEM (n = 4/group); **, P

    Journal: PLoS ONE

    Article Title: Mitogen- and Stress-Activated Kinase 1 (MSK1) Regulates Cigarette Smoke-Induced Histone Modifications on NF-?B-dependent Genes

    doi: 10.1371/journal.pone.0031378

    Figure Lengend Snippet: Cigarette smoke increased p-MSK1 (Thr581) and p-RelA/p65 (Ser276) in human bronchial and small airway epithelial cells as demonstrated by immunocytochemistry, and in mouse lung by immunoblotting. (A) Activation of MSK1 and RelA/p65 (Ser276) in H292 cells treated with CSE (1%) for 1 h. For immunocytochemistry, cells were fixed, permeabilized, and stained for the expression of p-MSK1 (Thr581), total MSK1, and phosphorylated RelA/p65 (Ser276) by immunofluorescence. Phosphorylated MSK1, total MSK1, and phosphorylated RelA/p65 is shown in red, and DNA (Hoechst nuclear staining) in blue, and merge represented as dark purple or pink. Results are representative cells from three separate experiments. The panel without primary antibodies was considered as negative control (NC). (B) Activation of MSK1 and RelA/p65 (Ser276) in SAEC cells treated with CSE (1%) for 1 h. For immunocytochemistry, cells were fixed, permeabilized, and stained for the expression of p-MSK1 (Thr581), total MSK1, and phosphorylated RelA/p65 (Ser276) by immunofluorescence. Phosphorylated MSK1, and phosphorylated RelA/p65 is shown in red, total MSK1 in green, and DNA (Hoechst nuclear staining) in blue, and merge represented as dark purple or pink. Results are representative cells from three separate experiments. The panel without primary antibodies was considered as negative control (NC). (C) Nuclear levels of MSK1 (phosphorylated and total MSK1) were increased in lung tissue of mouse exposed to CS determined by immunoblot analysis. Adult C57BL/6J mice were exposed to CS for 3 days and were sacrificed 24 hrs post-final CS exposure. Lamin B was used as protein loading control. The band intensity was measured by densitometry and data shown as fold change relative to Lamin B control. Data are shown as mean ± SEM (n = 4/group); **, P

    Article Snippet: MSK1, p-S276 (RelA/p65), RelA/p65, and Lamin B antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA).

    Techniques: Immunocytochemistry, Activation Assay, Staining, Expressing, Immunofluorescence, Negative Control, Mouse Assay

    NEK2 activates NF-κB signaling via PP1α/AKT. ( A ) USP7 was knocked down in ARP1 cells transduced with NEK2-OE after 72 hours induction with doxycycline (DOX). Nuclear and cytosolic fractionations were carried out. p65 levels were analyzed between EV and NEK2-OE with or without USP7 shRNA by Western blot. β-Actin and histone H3 (H3) were used as cytosolic and nuclear markers, respectively. ( B – D ) EV and NEK2-OE ARP1, OCI-MY5, and H1299 cells were lysed. NEK2, p65-S536 phosphorylation, IKK phosphorylation, and IκBα were analyzed by Western blot. ( E ) H1299 cells transiently transfected with EV or NEK2-OE (WT) or NEK2-K37R mutant (NEK2-Dead) were lysed, and NEK2 and p65-S536 phosphorylation was analyzed by Western blot. ( F ) ARP1 and OCI-MY5 cells transfected with EV or NEK2-OE were treated with vehicle or MK-2206 2HCl, an AKT inhibitor, for 30 minutes and then cells were lysed. p65-S536 phosphorylation was analyzed by Western blot. ( G ) NEK2-shRNA ARP1 cells were induced with DOX for 48 hours and then treated with tautomycin, a PP1α inhibitor, for another 24 hours. NEK2, p-p65-S536, p-PP1α, and p-AKT were analyzed by Western blot.

    Journal: The Journal of Clinical Investigation

    Article Title: Destabilizing NEK2 overcomes resistance to proteasome inhibition in multiple myeloma

    doi: 10.1172/JCI98765

    Figure Lengend Snippet: NEK2 activates NF-κB signaling via PP1α/AKT. ( A ) USP7 was knocked down in ARP1 cells transduced with NEK2-OE after 72 hours induction with doxycycline (DOX). Nuclear and cytosolic fractionations were carried out. p65 levels were analyzed between EV and NEK2-OE with or without USP7 shRNA by Western blot. β-Actin and histone H3 (H3) were used as cytosolic and nuclear markers, respectively. ( B – D ) EV and NEK2-OE ARP1, OCI-MY5, and H1299 cells were lysed. NEK2, p65-S536 phosphorylation, IKK phosphorylation, and IκBα were analyzed by Western blot. ( E ) H1299 cells transiently transfected with EV or NEK2-OE (WT) or NEK2-K37R mutant (NEK2-Dead) were lysed, and NEK2 and p65-S536 phosphorylation was analyzed by Western blot. ( F ) ARP1 and OCI-MY5 cells transfected with EV or NEK2-OE were treated with vehicle or MK-2206 2HCl, an AKT inhibitor, for 30 minutes and then cells were lysed. p65-S536 phosphorylation was analyzed by Western blot. ( G ) NEK2-shRNA ARP1 cells were induced with DOX for 48 hours and then treated with tautomycin, a PP1α inhibitor, for another 24 hours. NEK2, p-p65-S536, p-PP1α, and p-AKT were analyzed by Western blot.

    Article Snippet: Briefly, chromatin (5 μg) from the ARP1 myeloma cell line was used in ChIP assay using antibodies (4 μg) against NF-κB p65 (Santa Cruz Biotechnology, sc-372).

    Techniques: Transduction, shRNA, Western Blot, Transfection, Mutagenesis

    Working model of the interaction between NEK2 and USP7. USP7 binds to and stabilizes NEK2 by deubiquitination, allowing it to accumulate in myeloma cells. Accumulated NEK2 binds to and phosphorylates PP1α, resulting in loss its AKT-suppressing activity. Active AKT triggers the canonical NF-κB pathway by phosphorylating IKK, with subsequent phosphorylation and degradation of IκBα. p65 released from the complex with IκBα translocates into the nucleus, where it activates its target genes leading to drug resistance in myeloma. Ub, ubiquitin.

    Journal: The Journal of Clinical Investigation

    Article Title: Destabilizing NEK2 overcomes resistance to proteasome inhibition in multiple myeloma

    doi: 10.1172/JCI98765

    Figure Lengend Snippet: Working model of the interaction between NEK2 and USP7. USP7 binds to and stabilizes NEK2 by deubiquitination, allowing it to accumulate in myeloma cells. Accumulated NEK2 binds to and phosphorylates PP1α, resulting in loss its AKT-suppressing activity. Active AKT triggers the canonical NF-κB pathway by phosphorylating IKK, with subsequent phosphorylation and degradation of IκBα. p65 released from the complex with IκBα translocates into the nucleus, where it activates its target genes leading to drug resistance in myeloma. Ub, ubiquitin.

    Article Snippet: Briefly, chromatin (5 μg) from the ARP1 myeloma cell line was used in ChIP assay using antibodies (4 μg) against NF-κB p65 (Santa Cruz Biotechnology, sc-372).

    Techniques: Activity Assay

    INH1 and P5091 overcomes NEK2-induced bortezomib resistance. ( A ) Approximately 0.5 × 10 6 NEK2-OE ARP1 cells expressing luciferase were injected subcutaneously in both left and right flanks of NOD-Rag1 null mice. One week after injection of NEK2-OE cells, mice were treated with (i) vehicle, (ii) bortezomib (BTZ; 3 mg/kg, i.p., 2 times/week), (iii) INH1 (100 mg/kg, i.p., 3 times/week), (iv) P5091 (10 mg/kg, i.v., 2 times/week), (v) INH1 + BTZ, or (vii) P5091 + BTZ for 28 days. In vivo imaging showing the tumor growth in the different groups of mice before and after treatments at different time points. ( B ) Tumors from A were harvested and photographed. ( C ) Quantification of tumor volume from dissected tumors in B and Dunnett’s method was used to calculate the multiplicity-adjusted P values for each treatment and control group pair. *** P = 0.005; **** P = 0.0001. NS, no significance. ( D ) Quantification of tumor weight from dissected tumors in B and Dunnett’s method was used to calculate the multiplicity-adjusted P values for each treatment and control group pair. ** P = 0.0032; †† P = 0.0052; **** P = 0.0001. NS, no significance. ( E ) Tumors from B were lysed and analyzed by Western blot using NEK2, p-p65-S536, and GAPDH antibodies.

    Journal: The Journal of Clinical Investigation

    Article Title: Destabilizing NEK2 overcomes resistance to proteasome inhibition in multiple myeloma

    doi: 10.1172/JCI98765

    Figure Lengend Snippet: INH1 and P5091 overcomes NEK2-induced bortezomib resistance. ( A ) Approximately 0.5 × 10 6 NEK2-OE ARP1 cells expressing luciferase were injected subcutaneously in both left and right flanks of NOD-Rag1 null mice. One week after injection of NEK2-OE cells, mice were treated with (i) vehicle, (ii) bortezomib (BTZ; 3 mg/kg, i.p., 2 times/week), (iii) INH1 (100 mg/kg, i.p., 3 times/week), (iv) P5091 (10 mg/kg, i.v., 2 times/week), (v) INH1 + BTZ, or (vii) P5091 + BTZ for 28 days. In vivo imaging showing the tumor growth in the different groups of mice before and after treatments at different time points. ( B ) Tumors from A were harvested and photographed. ( C ) Quantification of tumor volume from dissected tumors in B and Dunnett’s method was used to calculate the multiplicity-adjusted P values for each treatment and control group pair. *** P = 0.005; **** P = 0.0001. NS, no significance. ( D ) Quantification of tumor weight from dissected tumors in B and Dunnett’s method was used to calculate the multiplicity-adjusted P values for each treatment and control group pair. ** P = 0.0032; †† P = 0.0052; **** P = 0.0001. NS, no significance. ( E ) Tumors from B were lysed and analyzed by Western blot using NEK2, p-p65-S536, and GAPDH antibodies.

    Article Snippet: Briefly, chromatin (5 μg) from the ARP1 myeloma cell line was used in ChIP assay using antibodies (4 μg) against NF-κB p65 (Santa Cruz Biotechnology, sc-372).

    Techniques: Expressing, Luciferase, Injection, Mouse Assay, In Vivo Imaging, Western Blot

    NEK2 activates the canonical NF-κB signaling pathway. ( A ) Affymetrix gene expression profiling data from 351 purified bone marrow plasma cell populations in the Total Therapy 2 (TT2) cohort was used to correlate the expression of NEK2 with the NF-κB signaling score. Pearson’s correlation was performed between log 2 [NEK2 signal] and the NF-κB signaling score. ( B ) H1299 cells were transfected with luciferase vector under a p65-dependent promoter, an internal Renilla control vector, p52 as a negative control, or a NEK2-OE vector. Results show that NEK2 overexpression increased the luciferase signal 2.5-fold compared with p52. ( C ) An unsupervised hierarchical cluster analysis was used to classify 351 multiple myeloma samples using NEK2 and 31 NF-κB genes regulated by NEK2. ( D – G ) Kaplan-Meier analyses show event-free survival (EFS) and overall survival (OS) of multiple myeloma patients enrolled in TT2 ( D and E ) and HOVON-65 ( F and G ) cohorts. Each line represents different subgroups identified in C and is described in the figure and color coded.

    Journal: The Journal of Clinical Investigation

    Article Title: Destabilizing NEK2 overcomes resistance to proteasome inhibition in multiple myeloma

    doi: 10.1172/JCI98765

    Figure Lengend Snippet: NEK2 activates the canonical NF-κB signaling pathway. ( A ) Affymetrix gene expression profiling data from 351 purified bone marrow plasma cell populations in the Total Therapy 2 (TT2) cohort was used to correlate the expression of NEK2 with the NF-κB signaling score. Pearson’s correlation was performed between log 2 [NEK2 signal] and the NF-κB signaling score. ( B ) H1299 cells were transfected with luciferase vector under a p65-dependent promoter, an internal Renilla control vector, p52 as a negative control, or a NEK2-OE vector. Results show that NEK2 overexpression increased the luciferase signal 2.5-fold compared with p52. ( C ) An unsupervised hierarchical cluster analysis was used to classify 351 multiple myeloma samples using NEK2 and 31 NF-κB genes regulated by NEK2. ( D – G ) Kaplan-Meier analyses show event-free survival (EFS) and overall survival (OS) of multiple myeloma patients enrolled in TT2 ( D and E ) and HOVON-65 ( F and G ) cohorts. Each line represents different subgroups identified in C and is described in the figure and color coded.

    Article Snippet: Briefly, chromatin (5 μg) from the ARP1 myeloma cell line was used in ChIP assay using antibodies (4 μg) against NF-κB p65 (Santa Cruz Biotechnology, sc-372).

    Techniques: Expressing, Purification, Transfection, Luciferase, Plasmid Preparation, Negative Control, Over Expression

    NEK2 activates the canonical NF-κB signaling pathway in primary multiple myeloma samples. ( A ) Primary myeloma samples from 16 patients (Pts) were lysed and analyzed by Western blot using NEK2, p-p65-S536, total p65 (p65), USP7, and GAPDH antibodies. ( B ) CD138-positive myeloma cells isolated from 4 primary myeloma patients were mounted on cytospin slides and analyzed by immunofluorescence using NEK2 and p-p65-S536 antibodies. DAPI staining was used to visualize nuclei. Yellow arrowheads indicate myeloma cells coexpressing NEK2 and p-p65-S536. Blue arrowheads show myeloma cells expressing p-p65-S536 with undetectable NEK2 levels. ( C ) EV and NEK2-OE ARP1 cells were treated with vehicle, BAY11-7082 (0.5 or 1.0 μM), and bortezomib (5 nM) alone or in combination. After 48 hours, cell viability was assessed by trypan blue staining and Dunnett’s method was used to calculate the multiplicity-adjusted P values for each treatment and control group pair. ** P = 0.0023; **** P = 0.0001. NS, no significance. Experiment was performed in triplicate. ( D ) to ubiquitinate NEK2 resulting in its stabilization.

    Journal: The Journal of Clinical Investigation

    Article Title: Destabilizing NEK2 overcomes resistance to proteasome inhibition in multiple myeloma

    doi: 10.1172/JCI98765

    Figure Lengend Snippet: NEK2 activates the canonical NF-κB signaling pathway in primary multiple myeloma samples. ( A ) Primary myeloma samples from 16 patients (Pts) were lysed and analyzed by Western blot using NEK2, p-p65-S536, total p65 (p65), USP7, and GAPDH antibodies. ( B ) CD138-positive myeloma cells isolated from 4 primary myeloma patients were mounted on cytospin slides and analyzed by immunofluorescence using NEK2 and p-p65-S536 antibodies. DAPI staining was used to visualize nuclei. Yellow arrowheads indicate myeloma cells coexpressing NEK2 and p-p65-S536. Blue arrowheads show myeloma cells expressing p-p65-S536 with undetectable NEK2 levels. ( C ) EV and NEK2-OE ARP1 cells were treated with vehicle, BAY11-7082 (0.5 or 1.0 μM), and bortezomib (5 nM) alone or in combination. After 48 hours, cell viability was assessed by trypan blue staining and Dunnett’s method was used to calculate the multiplicity-adjusted P values for each treatment and control group pair. ** P = 0.0023; **** P = 0.0001. NS, no significance. Experiment was performed in triplicate. ( D ) to ubiquitinate NEK2 resulting in its stabilization.

    Article Snippet: Briefly, chromatin (5 μg) from the ARP1 myeloma cell line was used in ChIP assay using antibodies (4 μg) against NF-κB p65 (Santa Cruz Biotechnology, sc-372).

    Techniques: Western Blot, Isolation, Immunofluorescence, Staining, Expressing

    NEK2 regulates heparanase expression via NF-κB. ( A ) Total RNA from EV or NEK2-OE or NEK2-shRNA ARP1 cells were harvested for gene expression profiling; NEK2 and HPSE mRNA levels are presented as bar graphs of duplicate samples. ( B ) The map of p65 binding to the HPSE promoter was obtained from the UCSC genome browser ChIP-seq data (track name: GM12878+TNFa RELA). HSPE contains 2 RELA (p65) binding sites across its sequence. H3K4me3 and H3K27Ac, typical for promoter and activator binding elements, respectively, show strong peaks at the p65 binding site of the HPSE sequence (red box). ( C ) ChIP-qPCR was performed in control cell (EV) and NEK2-OE ARP1 myeloma cells using p65 antibodies. The DNA occupancy of the HPSE promoter was analyzed by qPCR and a Student’s t test was performed to assess the significance. * P

    Journal: The Journal of Clinical Investigation

    Article Title: Destabilizing NEK2 overcomes resistance to proteasome inhibition in multiple myeloma

    doi: 10.1172/JCI98765

    Figure Lengend Snippet: NEK2 regulates heparanase expression via NF-κB. ( A ) Total RNA from EV or NEK2-OE or NEK2-shRNA ARP1 cells were harvested for gene expression profiling; NEK2 and HPSE mRNA levels are presented as bar graphs of duplicate samples. ( B ) The map of p65 binding to the HPSE promoter was obtained from the UCSC genome browser ChIP-seq data (track name: GM12878+TNFa RELA). HSPE contains 2 RELA (p65) binding sites across its sequence. H3K4me3 and H3K27Ac, typical for promoter and activator binding elements, respectively, show strong peaks at the p65 binding site of the HPSE sequence (red box). ( C ) ChIP-qPCR was performed in control cell (EV) and NEK2-OE ARP1 myeloma cells using p65 antibodies. The DNA occupancy of the HPSE promoter was analyzed by qPCR and a Student’s t test was performed to assess the significance. * P

    Article Snippet: Briefly, chromatin (5 μg) from the ARP1 myeloma cell line was used in ChIP assay using antibodies (4 μg) against NF-κB p65 (Santa Cruz Biotechnology, sc-372).

    Techniques: Expressing, shRNA, Binding Assay, Chromatin Immunoprecipitation, Sequencing, Real-time Polymerase Chain Reaction

    Targeting NEK2 overcomes acquired drug resistance in vivo. ( A ) The parental RPMI-8226 (8226) and drug-resistant RPMI-8226 (8226-DR) myeloma cells were treated with different bortezomib (BTZ) concentrations. Cell viability was analyzed by trypan blue staining after 48 hours. Viability experiments were performed in triplicate. ( B ) Western blots showing the expression of NEK2, p65, p-p65-S536, HPSE, and GAPDH in 8226 and 8226-DR myeloma cells. ( C ) Images of representative dissected tumors from mice treated with (i) vehicle, (ii) BTZ, (iii) INH1, (iv) P5091, (v) INH1 + BTZ, or (vi) P5091 + BTZ for 28 days. ( D ) Tumor volume was quantified from dissected tumors in C and Sidak’s method was used to calculate the multiplicity-adjusted P values for active treatment groups versus control. * P = 0.0172; *** P = 0.0004; ††† P = 0.0002; **** P

    Journal: The Journal of Clinical Investigation

    Article Title: Destabilizing NEK2 overcomes resistance to proteasome inhibition in multiple myeloma

    doi: 10.1172/JCI98765

    Figure Lengend Snippet: Targeting NEK2 overcomes acquired drug resistance in vivo. ( A ) The parental RPMI-8226 (8226) and drug-resistant RPMI-8226 (8226-DR) myeloma cells were treated with different bortezomib (BTZ) concentrations. Cell viability was analyzed by trypan blue staining after 48 hours. Viability experiments were performed in triplicate. ( B ) Western blots showing the expression of NEK2, p65, p-p65-S536, HPSE, and GAPDH in 8226 and 8226-DR myeloma cells. ( C ) Images of representative dissected tumors from mice treated with (i) vehicle, (ii) BTZ, (iii) INH1, (iv) P5091, (v) INH1 + BTZ, or (vi) P5091 + BTZ for 28 days. ( D ) Tumor volume was quantified from dissected tumors in C and Sidak’s method was used to calculate the multiplicity-adjusted P values for active treatment groups versus control. * P = 0.0172; *** P = 0.0004; ††† P = 0.0002; **** P

    Article Snippet: Briefly, chromatin (5 μg) from the ARP1 myeloma cell line was used in ChIP assay using antibodies (4 μg) against NF-κB p65 (Santa Cruz Biotechnology, sc-372).

    Techniques: In Vivo, Staining, Western Blot, Expressing, Mouse Assay

    NleC proteasomal-independent degradation of p65, p50, and IκBα. A , relative amount of NFκB luciferase reporter activity in pEGFP and pEGFP-NleC transfected cells treated, or not, with the proteasomal inhibitor MG132 revealing that the drug reduces basal NFκB activity and increases the inhibitory activity of NleC. Data shown are mean (±S.D.) of three experiments done in triplicate with level of significance (Student's t test) indicated. **, p ≤ 0.01; ***, p ≤ 0.005 as compared with pEGFP-transfected cells. B–D , representative immunoblots probing for p65, p50, IκB, pIκB (phosphorylated form targeted for proteasomal degradation), actin, and IKK(α and β) proteins in cellular extracts from cells transfected with pEGFP, pEGFP-NleC, or pEGFP-NleC 1–237 incubated, or not, with MG132. Data verify the inhibitory activity of MG132 on TNFα-treated cells (prevents proteasomal-dependent degradation of pIκB; see C ) and ability of eGFP-NleC, but not eGFP or eGFP-NleC 1–237 proteins, to deplete p50, p65, and/or IκBα from untreated and MG132-treated cells.

    Journal: The Journal of Biological Chemistry

    Article Title: Proteasome-independent Degradation of Canonical NF?B Complex Components by the NleC Protein of Pathogenic Escherichia coli *

    doi: 10.1074/jbc.M110.172254

    Figure Lengend Snippet: NleC proteasomal-independent degradation of p65, p50, and IκBα. A , relative amount of NFκB luciferase reporter activity in pEGFP and pEGFP-NleC transfected cells treated, or not, with the proteasomal inhibitor MG132 revealing that the drug reduces basal NFκB activity and increases the inhibitory activity of NleC. Data shown are mean (±S.D.) of three experiments done in triplicate with level of significance (Student's t test) indicated. **, p ≤ 0.01; ***, p ≤ 0.005 as compared with pEGFP-transfected cells. B–D , representative immunoblots probing for p65, p50, IκB, pIκB (phosphorylated form targeted for proteasomal degradation), actin, and IKK(α and β) proteins in cellular extracts from cells transfected with pEGFP, pEGFP-NleC, or pEGFP-NleC 1–237 incubated, or not, with MG132. Data verify the inhibitory activity of MG132 on TNFα-treated cells (prevents proteasomal-dependent degradation of pIκB; see C ) and ability of eGFP-NleC, but not eGFP or eGFP-NleC 1–237 proteins, to deplete p50, p65, and/or IκBα from untreated and MG132-treated cells.

    Article Snippet: Primary antibodies used were NFκB p65, NFκB p50, IκBα, IKKα/β (Santa Cruz Biotechnology), p65, phospho-p65 (Cell Signaling Technologies), actin (Sigma), and GFP (Zymed Laboratories Inc.).

    Techniques: Luciferase, Activity Assay, Transfection, Western Blot, Incubation

    Interaction of p65 and p50 with eGFP-NleC 1–237 and critical role for NleC residues 33–64. A , representative immunoblot of anti-GFP immunoprecipitate isolated from cells transfected with pEGFP, pEGFP-NleC, or pEGFP-NleC 1–237 probed for p65 and p50. The data reveals that p65 and p50 can be isolated with eGFP-NleC 1–237 but not eGFP or eGFP-NleC, the latter presumably due to rapid p65/p50 degradation. B , schematic of NleC N-terminal truncation variants constructed and screened in NFκB luciferase reporter assay. C , relative luciferase activity of cells expressing NleC and indicated variants, compared with control pEGFP-transfected cells. Data shown are mean (±S.D.) of three experiments done in triplicate with level of significance (Student's t test) indicated. *, p ≤ 0.05; ***, p ≤ 0.005 as compared with empty vector control. NleC 33–330 but not NleC 65–330 inhibits NFκB luciferase reporter activity revealing a critical role for residues between 33 and 64. D , representative immunoblot probed for GFP, p65, and actin that links loss of NFκB luciferase reporter activity of the NleC 65–330 variants with a major defect in depleting p65 from the cell.

    Journal: The Journal of Biological Chemistry

    Article Title: Proteasome-independent Degradation of Canonical NF?B Complex Components by the NleC Protein of Pathogenic Escherichia coli *

    doi: 10.1074/jbc.M110.172254

    Figure Lengend Snippet: Interaction of p65 and p50 with eGFP-NleC 1–237 and critical role for NleC residues 33–64. A , representative immunoblot of anti-GFP immunoprecipitate isolated from cells transfected with pEGFP, pEGFP-NleC, or pEGFP-NleC 1–237 probed for p65 and p50. The data reveals that p65 and p50 can be isolated with eGFP-NleC 1–237 but not eGFP or eGFP-NleC, the latter presumably due to rapid p65/p50 degradation. B , schematic of NleC N-terminal truncation variants constructed and screened in NFκB luciferase reporter assay. C , relative luciferase activity of cells expressing NleC and indicated variants, compared with control pEGFP-transfected cells. Data shown are mean (±S.D.) of three experiments done in triplicate with level of significance (Student's t test) indicated. *, p ≤ 0.05; ***, p ≤ 0.005 as compared with empty vector control. NleC 33–330 but not NleC 65–330 inhibits NFκB luciferase reporter activity revealing a critical role for residues between 33 and 64. D , representative immunoblot probed for GFP, p65, and actin that links loss of NFκB luciferase reporter activity of the NleC 65–330 variants with a major defect in depleting p65 from the cell.

    Article Snippet: Primary antibodies used were NFκB p65, NFκB p50, IκBα, IKKα/β (Santa Cruz Biotechnology), p65, phospho-p65 (Cell Signaling Technologies), actin (Sigma), and GFP (Zymed Laboratories Inc.).

    Techniques: Isolation, Transfection, Construct, Luciferase, Reporter Assay, Activity Assay, Expressing, Plasmid Preparation

    NleC inhibits NFκB function by targeting is constituents. Fold increase in NFκB-dependent luciferase activity of Hela cells co-transfected with vectors encoding pEGFP or pEGFP-NleC and NFκB pathway components TRAF2, IKKα, or IKKβ ( A ) and p65 ( C ). Transfection with pEGFP-NleC inhibits luciferase activity associated with plasmid expression of all interrogated IKK pathway components. Data shown are mean (±S.D.) of three experiments done in triplicate with level of significance (Student's t test) indicated. ***, p ≤ 0.005 as compared with empty vector control. B , representative immunoblot probing for actin, IKK (α and β), p65, and p50 demonstrates similar levels of IKK and loading control, actin, with decreased cellular levels of p65 and p50 in pEGFP-NleC-transfected cells.

    Journal: The Journal of Biological Chemistry

    Article Title: Proteasome-independent Degradation of Canonical NF?B Complex Components by the NleC Protein of Pathogenic Escherichia coli *

    doi: 10.1074/jbc.M110.172254

    Figure Lengend Snippet: NleC inhibits NFκB function by targeting is constituents. Fold increase in NFκB-dependent luciferase activity of Hela cells co-transfected with vectors encoding pEGFP or pEGFP-NleC and NFκB pathway components TRAF2, IKKα, or IKKβ ( A ) and p65 ( C ). Transfection with pEGFP-NleC inhibits luciferase activity associated with plasmid expression of all interrogated IKK pathway components. Data shown are mean (±S.D.) of three experiments done in triplicate with level of significance (Student's t test) indicated. ***, p ≤ 0.005 as compared with empty vector control. B , representative immunoblot probing for actin, IKK (α and β), p65, and p50 demonstrates similar levels of IKK and loading control, actin, with decreased cellular levels of p65 and p50 in pEGFP-NleC-transfected cells.

    Article Snippet: Primary antibodies used were NFκB p65, NFκB p50, IκBα, IKKα/β (Santa Cruz Biotechnology), p65, phospho-p65 (Cell Signaling Technologies), actin (Sigma), and GFP (Zymed Laboratories Inc.).

    Techniques: Luciferase, Activity Assay, Transfection, Plasmid Preparation, Expressing

    Model for NleC inhibition of NFκB activity. NleC residues 1–66 are sufficient to, directly or indirectly, recruit p50 and p65 to degrade these proteins as well as upstream IκB, but not IKK or TRAF2, proteins by a process inhibited by adding metalloprotease inhibitors (EDTA and GM6001) or disrupting the consensus zinc metalloprotease motif (HE XX H) encompassing residues 183–187.

    Journal: The Journal of Biological Chemistry

    Article Title: Proteasome-independent Degradation of Canonical NF?B Complex Components by the NleC Protein of Pathogenic Escherichia coli *

    doi: 10.1074/jbc.M110.172254

    Figure Lengend Snippet: Model for NleC inhibition of NFκB activity. NleC residues 1–66 are sufficient to, directly or indirectly, recruit p50 and p65 to degrade these proteins as well as upstream IκB, but not IKK or TRAF2, proteins by a process inhibited by adding metalloprotease inhibitors (EDTA and GM6001) or disrupting the consensus zinc metalloprotease motif (HE XX H) encompassing residues 183–187.

    Article Snippet: Primary antibodies used were NFκB p65, NFκB p50, IκBα, IKKα/β (Santa Cruz Biotechnology), p65, phospho-p65 (Cell Signaling Technologies), actin (Sigma), and GFP (Zymed Laboratories Inc.).

    Techniques: Inhibition, Activity Assay

    Metalloprotease inhibitors and disruption of the consensus zinc metalloprotease motif of NleC abolish p65 and p50 degradation. A , representative immunoblot of cellular extracts from pEGFP, pEGFP-NleC, and pEGFP-NleC transfected treated, or not, with metalloprotease inhibitors (EDTA or GM6001). B , relative amount of NFκB luciferase reporter activity for cells described in A. C , representative immunoblot demonstrating co-immunoprecipitating of p65 and p50 with eGFP-NleC 1–66 , but not eGFP-NleC 65–330 , with EDTA-mediated inhibition of protease activity revealing that full length NleC interacts with p65 and p50. The consensus zinc metalloprotease motif of NleC and destructive substitutions ( D ) with immunoblot ( E ) and NFκB luciferase reporter activity studies revealing the key role for this feature in the cellular loss of p50/p65 and NFκB activity ( F ), respectively, are shown. Luciferase data are mean (±S.D.) of three experiments done in triplicate with level of significance (Student's t test) indicated. ***, p ≤ 0.005 as compared with empty vector control.

    Journal: The Journal of Biological Chemistry

    Article Title: Proteasome-independent Degradation of Canonical NF?B Complex Components by the NleC Protein of Pathogenic Escherichia coli *

    doi: 10.1074/jbc.M110.172254

    Figure Lengend Snippet: Metalloprotease inhibitors and disruption of the consensus zinc metalloprotease motif of NleC abolish p65 and p50 degradation. A , representative immunoblot of cellular extracts from pEGFP, pEGFP-NleC, and pEGFP-NleC transfected treated, or not, with metalloprotease inhibitors (EDTA or GM6001). B , relative amount of NFκB luciferase reporter activity for cells described in A. C , representative immunoblot demonstrating co-immunoprecipitating of p65 and p50 with eGFP-NleC 1–66 , but not eGFP-NleC 65–330 , with EDTA-mediated inhibition of protease activity revealing that full length NleC interacts with p65 and p50. The consensus zinc metalloprotease motif of NleC and destructive substitutions ( D ) with immunoblot ( E ) and NFκB luciferase reporter activity studies revealing the key role for this feature in the cellular loss of p50/p65 and NFκB activity ( F ), respectively, are shown. Luciferase data are mean (±S.D.) of three experiments done in triplicate with level of significance (Student's t test) indicated. ***, p ≤ 0.005 as compared with empty vector control.

    Article Snippet: Primary antibodies used were NFκB p65, NFκB p50, IκBα, IKKα/β (Santa Cruz Biotechnology), p65, phospho-p65 (Cell Signaling Technologies), actin (Sigma), and GFP (Zymed Laboratories Inc.).

    Techniques: Transfection, Luciferase, Activity Assay, Inhibition, Plasmid Preparation

    Key role for NleC residues 237–266 in inducing cellular loss of p65 and p50. A , schematic of NleC C-terminal truncation variants constructed and screened in the NFκB luciferase reporter assay. B , relative luciferase activity of cells expressing NleC, and variants thereof, relative to pEGFP-transfected cells. NleC 1–266 , but not NleC 1–237 , inhibits NFκB luciferase reporter activity revealing a critical role for residues between 237–266. Data shown are mean (±S.D.) of three experiments done in triplicate with level of significance (Student's t test) indicated. *, p ≤ 0.05; ***, p ≤ 0.005 as compared with empty vector control. C , representative immunoblot probed for p65 (and phosphorylated activation-associated form; pp65), p50, IKK (α and β), actin, and GFP that links loss of NFκB luciferase reporter activity with an inability to deplete p65 and p50 from HeLa cells. aa , amino acids.

    Journal: The Journal of Biological Chemistry

    Article Title: Proteasome-independent Degradation of Canonical NF?B Complex Components by the NleC Protein of Pathogenic Escherichia coli *

    doi: 10.1074/jbc.M110.172254

    Figure Lengend Snippet: Key role for NleC residues 237–266 in inducing cellular loss of p65 and p50. A , schematic of NleC C-terminal truncation variants constructed and screened in the NFκB luciferase reporter assay. B , relative luciferase activity of cells expressing NleC, and variants thereof, relative to pEGFP-transfected cells. NleC 1–266 , but not NleC 1–237 , inhibits NFκB luciferase reporter activity revealing a critical role for residues between 237–266. Data shown are mean (±S.D.) of three experiments done in triplicate with level of significance (Student's t test) indicated. *, p ≤ 0.05; ***, p ≤ 0.005 as compared with empty vector control. C , representative immunoblot probed for p65 (and phosphorylated activation-associated form; pp65), p50, IKK (α and β), actin, and GFP that links loss of NFκB luciferase reporter activity with an inability to deplete p65 and p50 from HeLa cells. aa , amino acids.

    Article Snippet: Primary antibodies used were NFκB p65, NFκB p50, IκBα, IKKα/β (Santa Cruz Biotechnology), p65, phospho-p65 (Cell Signaling Technologies), actin (Sigma), and GFP (Zymed Laboratories Inc.).

    Techniques: Construct, Luciferase, Reporter Assay, Activity Assay, Expressing, Transfection, Plasmid Preparation, Activation Assay

    Quantification of IL‐1α‐mediated enhancer modifications and p65 NF‐κB binding in the human IL8 and CXCL2 chemokine loci Published ChIP‐seq data from KB cells (Jurida et al , 2015 ; GSE64224 and GSE52470) were used to annotate active enhancers and p65 NF‐κB recruitment in untreated cells compared to cells stimulated with IL‐1α for 60 min ± TAKi. The browser views show ChIP‐seq profiles for all conditions of the IL8 and CXCL2 chemokine loci. Gray areas highlight four chromatin regions with IL‐1α‐inducible H3K27 acetylation and p65 binding. As indicated by black horizontal bars, these chromatin regions were provisionally designated by us as “class II enhancers” (Jurida et al , 2015 ) to distinguish them from the entire repertoire of all active (i.e., H3K4me1‐ and H3K27ac‐positive) enhancers. Vertical bars indicate predicted NF‐κB motifs in the DNA sequence and the positions of all sgRNAs used in this study for CRISPRa (see Fig 6 ) or for enhancer or promoter deletions (see Figs 3 and EV4). Bar graphs show cumulative read counts across the four IL‐1α‐regulated and TAKi‐sensitive class II enhancers. The two flanking enhancers with strongest p65 binding (2 and 4) are the ones investigated in detail in this study.

    Journal: The EMBO Journal

    Article Title: Distinct IL‐1α‐responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

    doi: 10.15252/embj.2019101533

    Figure Lengend Snippet: Quantification of IL‐1α‐mediated enhancer modifications and p65 NF‐κB binding in the human IL8 and CXCL2 chemokine loci Published ChIP‐seq data from KB cells (Jurida et al , 2015 ; GSE64224 and GSE52470) were used to annotate active enhancers and p65 NF‐κB recruitment in untreated cells compared to cells stimulated with IL‐1α for 60 min ± TAKi. The browser views show ChIP‐seq profiles for all conditions of the IL8 and CXCL2 chemokine loci. Gray areas highlight four chromatin regions with IL‐1α‐inducible H3K27 acetylation and p65 binding. As indicated by black horizontal bars, these chromatin regions were provisionally designated by us as “class II enhancers” (Jurida et al , 2015 ) to distinguish them from the entire repertoire of all active (i.e., H3K4me1‐ and H3K27ac‐positive) enhancers. Vertical bars indicate predicted NF‐κB motifs in the DNA sequence and the positions of all sgRNAs used in this study for CRISPRa (see Fig 6 ) or for enhancer or promoter deletions (see Figs 3 and EV4). Bar graphs show cumulative read counts across the four IL‐1α‐regulated and TAKi‐sensitive class II enhancers. The two flanking enhancers with strongest p65 binding (2 and 4) are the ones investigated in detail in this study.

    Article Snippet: Primary antibodies against the following proteins or peptides were used: anti‐β‐actin (Santa Cruz, #sc‐4778), anti‐CRISPR‐Cas9 (Abcam, #191468), anti‐CTCF (Millipore, #07‐729), anti‐FLAG (Sigma‐Aldrich, #F1804), anti‐H3 (Abcam, #ab1791), anti‐H3K4me1 (Abcam, #ab8895), anti‐H3K27ac (Diagenode, Pab‐174‐050), anti‐H3K27me3 (Millipore, #07‐449), anti‐H3K36ac (Diagenode, #C15410307), anti‐(PS32)‐IκBα (Cell Signaling, #2859), anti‐IκBα (Cell Signaling, #9242), anti‐P(T183/Y185)‐JNK (Cell Signaling, #9251), anti‐JNK (Santa Cruz, #sc‐571), anti‐P(S536)‐p65 (Cell Signaling, #3033), anti‐p65 (Santa Cruz, #sc‐372; #sc‐8008), anti‐P(T180/Y182)‐p38 (Zymed, #36‐8500), anti‐p38 (Cell Signaling, #9212), anti‐puromycin (3RH11; Kerafast, #EQ0001), anti‐P(S2)‐RNA Pol II (Abcam, #ab5095), anti‐P(S5)‐Pol II (Abcam, #ab5131), anti‐RNA‐Pol II (Millipore, #17‐620), anti‐tubulin (Santa Cruz, #sc‐8035), and normal rabbit IgG (Santa Cruz, #sc‐2027; Cell Signaling #2729).

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Sequencing

    Spatial chromatin interactions in the IL8 locus are rewired by deleting p65‐binding cis ‐elements within enhancers i4C profiles in the 1 Mbp around the IL8 locus on chromosome 4 ( ideogram ) from control (empty vector) and enhancer‐mutant (Δp65 eIL8 and Δp65 eCXCL2 ) HeLa lines ± IL‐1α stimulation for 60 min, generated using the IL8 promoter ( pink highlight ) or enhancer ( blue highlight ) as a viewpoint. For the IL8 promoter, the average of two biological replicates is shown, while for the IL8 enhancer, data from one replicate are shown. Below each profile, significantly strong ( brown ), medium ( red ), or weaker interactions ( orange ) called via foursig are indicated. All profiles are shown aligned to gene models ( blue ) and to CTCF, H3K4me1, H3K4me3, H3K27ac, and RNA polymerase II ENCODE HeLa‐S3 ChIP‐seq profiles. The breadth of TADs in the locus is indicated above. Meta‐plots showing coverage of H3K27ac ChIP‐seq signal at i4C fragments ± 1 kbp contacted by the IL8 promoter or enhancer in control cells (empty vector) in the presence ( magenta ) or absence ( gray ) of IL‐1α stimulation for 60 min, and in enhancer‐mutant cells (Δp65 eIL8 , blue ; Δp65 eCXCL2 , green ) after IL‐1α stimulation. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Distinct IL‐1α‐responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

    doi: 10.15252/embj.2019101533

    Figure Lengend Snippet: Spatial chromatin interactions in the IL8 locus are rewired by deleting p65‐binding cis ‐elements within enhancers i4C profiles in the 1 Mbp around the IL8 locus on chromosome 4 ( ideogram ) from control (empty vector) and enhancer‐mutant (Δp65 eIL8 and Δp65 eCXCL2 ) HeLa lines ± IL‐1α stimulation for 60 min, generated using the IL8 promoter ( pink highlight ) or enhancer ( blue highlight ) as a viewpoint. For the IL8 promoter, the average of two biological replicates is shown, while for the IL8 enhancer, data from one replicate are shown. Below each profile, significantly strong ( brown ), medium ( red ), or weaker interactions ( orange ) called via foursig are indicated. All profiles are shown aligned to gene models ( blue ) and to CTCF, H3K4me1, H3K4me3, H3K27ac, and RNA polymerase II ENCODE HeLa‐S3 ChIP‐seq profiles. The breadth of TADs in the locus is indicated above. Meta‐plots showing coverage of H3K27ac ChIP‐seq signal at i4C fragments ± 1 kbp contacted by the IL8 promoter or enhancer in control cells (empty vector) in the presence ( magenta ) or absence ( gray ) of IL‐1α stimulation for 60 min, and in enhancer‐mutant cells (Δp65 eIL8 , blue ; Δp65 eCXCL2 , green ) after IL‐1α stimulation. Source data are available online for this figure.

    Article Snippet: Primary antibodies against the following proteins or peptides were used: anti‐β‐actin (Santa Cruz, #sc‐4778), anti‐CRISPR‐Cas9 (Abcam, #191468), anti‐CTCF (Millipore, #07‐729), anti‐FLAG (Sigma‐Aldrich, #F1804), anti‐H3 (Abcam, #ab1791), anti‐H3K4me1 (Abcam, #ab8895), anti‐H3K27ac (Diagenode, Pab‐174‐050), anti‐H3K27me3 (Millipore, #07‐449), anti‐H3K36ac (Diagenode, #C15410307), anti‐(PS32)‐IκBα (Cell Signaling, #2859), anti‐IκBα (Cell Signaling, #9242), anti‐P(T183/Y185)‐JNK (Cell Signaling, #9251), anti‐JNK (Santa Cruz, #sc‐571), anti‐P(S536)‐p65 (Cell Signaling, #3033), anti‐p65 (Santa Cruz, #sc‐372; #sc‐8008), anti‐P(T180/Y182)‐p38 (Zymed, #36‐8500), anti‐p38 (Cell Signaling, #9212), anti‐puromycin (3RH11; Kerafast, #EQ0001), anti‐P(S2)‐RNA Pol II (Abcam, #ab5095), anti‐P(S5)‐Pol II (Abcam, #ab5131), anti‐RNA‐Pol II (Millipore, #17‐620), anti‐tubulin (Santa Cruz, #sc‐8035), and normal rabbit IgG (Santa Cruz, #sc‐2027; Cell Signaling #2729).

    Techniques: Binding Assay, Plasmid Preparation, Mutagenesis, Generated, Chromatin Immunoprecipitation

    Deletion of the p65‐binding site from the IL8 promoter only affects IL8 expression Genome browser view of the IL8 chemokine locus on human chromosome 4 showing H3K4me1, H3K4me3, H3K27ac, and RNA polymerase II ENCODE ChIP‐seq profiles from HeLa‐S3 cells relative to the IL8 gene model ( blue ). The location of the deleted NF‐κB binding site in the IL8 promoter is indicated ( orange ), and Sanger sequencing of PCR amplicons generated from genomic DNA of the Δp65 pIL8 cell line shows removal of 57 bp. Immunoblot analysis of extracts from parental HeLa cells (wt), vector controls, or Δp65 pIL8 cells was compared to cells stably expressing a sgRNA targeting the RELA (Δ RELA ) gene which encodes for the p65 NF‐κB subunit in the presence (+) or absence (−) of IL‐1α stimulation for 30 min or 60 min. Antibodies against p65 confirm the strong suppression of p65 in Δ RELA cells. Antibodies against P(S536)‐p65, P‐IκBα, and IκBα reveal normal IL‐1α‐mediated signaling in Δp65 pIL8 cells compared to control cells, while Δ RELA cells show reduced levels of the IL‐1α and the p65 target gene IκBα. β‐Actin levels provide a loading control. Cas9 antibodies reveal the levels of FLAG‐Cas9 present in the stable cell cultures. mRNA levels of seven IL‐1α‐responsive genes in parental (wt), control (empty vector), or promoter‐mutant (Δp65 pIL8 ) HeLa lines were assessed by RT–qPCR (mean levels ± SEM, n = 4) at 60 min after IL‐1α stimulation. Asterisks show significanc e of changes in basal and IL‐1α‐stimulated conditions compared to the corresponding vector control samples ( P

    Journal: The EMBO Journal

    Article Title: Distinct IL‐1α‐responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

    doi: 10.15252/embj.2019101533

    Figure Lengend Snippet: Deletion of the p65‐binding site from the IL8 promoter only affects IL8 expression Genome browser view of the IL8 chemokine locus on human chromosome 4 showing H3K4me1, H3K4me3, H3K27ac, and RNA polymerase II ENCODE ChIP‐seq profiles from HeLa‐S3 cells relative to the IL8 gene model ( blue ). The location of the deleted NF‐κB binding site in the IL8 promoter is indicated ( orange ), and Sanger sequencing of PCR amplicons generated from genomic DNA of the Δp65 pIL8 cell line shows removal of 57 bp. Immunoblot analysis of extracts from parental HeLa cells (wt), vector controls, or Δp65 pIL8 cells was compared to cells stably expressing a sgRNA targeting the RELA (Δ RELA ) gene which encodes for the p65 NF‐κB subunit in the presence (+) or absence (−) of IL‐1α stimulation for 30 min or 60 min. Antibodies against p65 confirm the strong suppression of p65 in Δ RELA cells. Antibodies against P(S536)‐p65, P‐IκBα, and IκBα reveal normal IL‐1α‐mediated signaling in Δp65 pIL8 cells compared to control cells, while Δ RELA cells show reduced levels of the IL‐1α and the p65 target gene IκBα. β‐Actin levels provide a loading control. Cas9 antibodies reveal the levels of FLAG‐Cas9 present in the stable cell cultures. mRNA levels of seven IL‐1α‐responsive genes in parental (wt), control (empty vector), or promoter‐mutant (Δp65 pIL8 ) HeLa lines were assessed by RT–qPCR (mean levels ± SEM, n = 4) at 60 min after IL‐1α stimulation. Asterisks show significanc e of changes in basal and IL‐1α‐stimulated conditions compared to the corresponding vector control samples ( P

    Article Snippet: Primary antibodies against the following proteins or peptides were used: anti‐β‐actin (Santa Cruz, #sc‐4778), anti‐CRISPR‐Cas9 (Abcam, #191468), anti‐CTCF (Millipore, #07‐729), anti‐FLAG (Sigma‐Aldrich, #F1804), anti‐H3 (Abcam, #ab1791), anti‐H3K4me1 (Abcam, #ab8895), anti‐H3K27ac (Diagenode, Pab‐174‐050), anti‐H3K27me3 (Millipore, #07‐449), anti‐H3K36ac (Diagenode, #C15410307), anti‐(PS32)‐IκBα (Cell Signaling, #2859), anti‐IκBα (Cell Signaling, #9242), anti‐P(T183/Y185)‐JNK (Cell Signaling, #9251), anti‐JNK (Santa Cruz, #sc‐571), anti‐P(S536)‐p65 (Cell Signaling, #3033), anti‐p65 (Santa Cruz, #sc‐372; #sc‐8008), anti‐P(T180/Y182)‐p38 (Zymed, #36‐8500), anti‐p38 (Cell Signaling, #9212), anti‐puromycin (3RH11; Kerafast, #EQ0001), anti‐P(S2)‐RNA Pol II (Abcam, #ab5095), anti‐P(S5)‐Pol II (Abcam, #ab5131), anti‐RNA‐Pol II (Millipore, #17‐620), anti‐tubulin (Santa Cruz, #sc‐8035), and normal rabbit IgG (Santa Cruz, #sc‐2027; Cell Signaling #2729).

    Techniques: Binding Assay, Expressing, Chromatin Immunoprecipitation, Sequencing, Polymerase Chain Reaction, Generated, Plasmid Preparation, Stable Transfection, Mutagenesis, Quantitative RT-PCR

    The IL‐1α–TAK1 pathway regulates spatial chromatin interactions by the CXCL2 locus Cross‐linking‐free chromosome conformation capture (i4C) analysis was performed using chromatin from KB cells ± IL‐1α stimulation for 60 min in the presence or absence of a TAK inhibitor (TAKi). Shown are i4C profiles in the 1 Mbp around the CXCL2 locus on chromosome 4 ( ideogram ). Average read counts of two biological replicates are plotted, generated using the CXCL2 promoter ( blue highlight ) or enhancer ( pink highlight ) as a viewpoint. The region of the IL8 promoter/enhancer is also shown ( gray highlight ). Below each profile, significantly strong ( brown ), medium ( red ), or weaker interactions ( orange ) called via foursig software (Williams et al , 2014 ) are indicated. All profiles are shown aligned to gene models ( blue ) and CTCF ChIP‐seq, as well as to H3K27ac and H3K4me1 ChIP‐seq data from KB cells (GSE64224 + GSE52470) performed under the same conditions (Jurida et al , 2015 ). The breadth of topologically associating domains (TADs) in the locus is indicated above. Meta‐plots showing coverage of ATAC‐seq (this study) and H3K27ac, p65, and RNA polymerase II (RNAPII) ChIP‐seq signals (GSE64224 + GSE52470) at i4C fragments ± 1 kbp contacted by the CXCL2 promoter or enhancer in KB cells ± IL‐1α stimulation for 60 min in the presence or absence of a TAK inhibitor (TAKi). Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Distinct IL‐1α‐responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

    doi: 10.15252/embj.2019101533

    Figure Lengend Snippet: The IL‐1α–TAK1 pathway regulates spatial chromatin interactions by the CXCL2 locus Cross‐linking‐free chromosome conformation capture (i4C) analysis was performed using chromatin from KB cells ± IL‐1α stimulation for 60 min in the presence or absence of a TAK inhibitor (TAKi). Shown are i4C profiles in the 1 Mbp around the CXCL2 locus on chromosome 4 ( ideogram ). Average read counts of two biological replicates are plotted, generated using the CXCL2 promoter ( blue highlight ) or enhancer ( pink highlight ) as a viewpoint. The region of the IL8 promoter/enhancer is also shown ( gray highlight ). Below each profile, significantly strong ( brown ), medium ( red ), or weaker interactions ( orange ) called via foursig software (Williams et al , 2014 ) are indicated. All profiles are shown aligned to gene models ( blue ) and CTCF ChIP‐seq, as well as to H3K27ac and H3K4me1 ChIP‐seq data from KB cells (GSE64224 + GSE52470) performed under the same conditions (Jurida et al , 2015 ). The breadth of topologically associating domains (TADs) in the locus is indicated above. Meta‐plots showing coverage of ATAC‐seq (this study) and H3K27ac, p65, and RNA polymerase II (RNAPII) ChIP‐seq signals (GSE64224 + GSE52470) at i4C fragments ± 1 kbp contacted by the CXCL2 promoter or enhancer in KB cells ± IL‐1α stimulation for 60 min in the presence or absence of a TAK inhibitor (TAKi). Source data are available online for this figure.

    Article Snippet: Primary antibodies against the following proteins or peptides were used: anti‐β‐actin (Santa Cruz, #sc‐4778), anti‐CRISPR‐Cas9 (Abcam, #191468), anti‐CTCF (Millipore, #07‐729), anti‐FLAG (Sigma‐Aldrich, #F1804), anti‐H3 (Abcam, #ab1791), anti‐H3K4me1 (Abcam, #ab8895), anti‐H3K27ac (Diagenode, Pab‐174‐050), anti‐H3K27me3 (Millipore, #07‐449), anti‐H3K36ac (Diagenode, #C15410307), anti‐(PS32)‐IκBα (Cell Signaling, #2859), anti‐IκBα (Cell Signaling, #9242), anti‐P(T183/Y185)‐JNK (Cell Signaling, #9251), anti‐JNK (Santa Cruz, #sc‐571), anti‐P(S536)‐p65 (Cell Signaling, #3033), anti‐p65 (Santa Cruz, #sc‐372; #sc‐8008), anti‐P(T180/Y182)‐p38 (Zymed, #36‐8500), anti‐p38 (Cell Signaling, #9212), anti‐puromycin (3RH11; Kerafast, #EQ0001), anti‐P(S2)‐RNA Pol II (Abcam, #ab5095), anti‐P(S5)‐Pol II (Abcam, #ab5131), anti‐RNA‐Pol II (Millipore, #17‐620), anti‐tubulin (Santa Cruz, #sc‐8035), and normal rabbit IgG (Santa Cruz, #sc‐2027; Cell Signaling #2729).

    Techniques: Generated, Software, Chromatin Immunoprecipitation

    Differential regulation of enhancers and promoters of IL‐1α target loci NF‐κB (p65), histone marks, and RNA polymerase II enrichment at the IL8 and CXCL2 promoter and enhancer in control (empty vector) or mutated (Δp65 eIL8 and Δp65 eCXCL2 ) HeLa lines were assessed by ChIP‐qPCR (mean enrichment over input ± SEM) at the indicated times after IL‐1α stimulation. IgG, H3K27ac, H3K4me1, H3, and p65 ChIP‐qPCR data are from three (vector, Δp65 eCXCL2 ) or four (Δp65 EIL8 ) independent experiments performed in duplicate; all others are from at least two independent experiments. *: significantly different to vector controls; P

    Journal: The EMBO Journal

    Article Title: Distinct IL‐1α‐responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

    doi: 10.15252/embj.2019101533

    Figure Lengend Snippet: Differential regulation of enhancers and promoters of IL‐1α target loci NF‐κB (p65), histone marks, and RNA polymerase II enrichment at the IL8 and CXCL2 promoter and enhancer in control (empty vector) or mutated (Δp65 eIL8 and Δp65 eCXCL2 ) HeLa lines were assessed by ChIP‐qPCR (mean enrichment over input ± SEM) at the indicated times after IL‐1α stimulation. IgG, H3K27ac, H3K4me1, H3, and p65 ChIP‐qPCR data are from three (vector, Δp65 eCXCL2 ) or four (Δp65 EIL8 ) independent experiments performed in duplicate; all others are from at least two independent experiments. *: significantly different to vector controls; P

    Article Snippet: Primary antibodies against the following proteins or peptides were used: anti‐β‐actin (Santa Cruz, #sc‐4778), anti‐CRISPR‐Cas9 (Abcam, #191468), anti‐CTCF (Millipore, #07‐729), anti‐FLAG (Sigma‐Aldrich, #F1804), anti‐H3 (Abcam, #ab1791), anti‐H3K4me1 (Abcam, #ab8895), anti‐H3K27ac (Diagenode, Pab‐174‐050), anti‐H3K27me3 (Millipore, #07‐449), anti‐H3K36ac (Diagenode, #C15410307), anti‐(PS32)‐IκBα (Cell Signaling, #2859), anti‐IκBα (Cell Signaling, #9242), anti‐P(T183/Y185)‐JNK (Cell Signaling, #9251), anti‐JNK (Santa Cruz, #sc‐571), anti‐P(S536)‐p65 (Cell Signaling, #3033), anti‐p65 (Santa Cruz, #sc‐372; #sc‐8008), anti‐P(T180/Y182)‐p38 (Zymed, #36‐8500), anti‐p38 (Cell Signaling, #9212), anti‐puromycin (3RH11; Kerafast, #EQ0001), anti‐P(S2)‐RNA Pol II (Abcam, #ab5095), anti‐P(S5)‐Pol II (Abcam, #ab5131), anti‐RNA‐Pol II (Millipore, #17‐620), anti‐tubulin (Santa Cruz, #sc‐8035), and normal rabbit IgG (Santa Cruz, #sc‐2027; Cell Signaling #2729).

    Techniques: Plasmid Preparation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Analyses of mRNA stabilities and the global IL‐1α gene expression response in enhancer‐mutant HeLa Empty vector controls or Δp65 eIL8 , Δp65 eCXCL2 , and Δp65 eIL8+eCXCL2 enhancer‐mutant HeLa lines were stimulated with IL‐1α for 60 min or were left untreated. Then, actinomycin D (5 μg/ml) was added to stop transcription and RNAs were immediately extracted (reference time point 0 min) or incubations were continued to monitor the decay of IL8 and CXCL2 mRNAs at different time points. RT–qPCR was performed from total RNA, and changes in mRNA expression were calculated and are depicted relative to the 0‐min time point (mean levels ± SEM, normalized to GUSB ; n = 2). Summary of microarray gene expression analysis performed in HeLa cells ± IL‐1α stimulation for 60 min on control (empty vector; n = 4) and three p65 enhancer‐deletion lines (Δp65 eIL8 / ‐ CXCL2 / ‐ IL8+CXCL2 ; n = 2). Differentially expressed genes were identified based on a moderated t ‐test ( P‐ value

    Journal: The EMBO Journal

    Article Title: Distinct IL‐1α‐responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

    doi: 10.15252/embj.2019101533

    Figure Lengend Snippet: Analyses of mRNA stabilities and the global IL‐1α gene expression response in enhancer‐mutant HeLa Empty vector controls or Δp65 eIL8 , Δp65 eCXCL2 , and Δp65 eIL8+eCXCL2 enhancer‐mutant HeLa lines were stimulated with IL‐1α for 60 min or were left untreated. Then, actinomycin D (5 μg/ml) was added to stop transcription and RNAs were immediately extracted (reference time point 0 min) or incubations were continued to monitor the decay of IL8 and CXCL2 mRNAs at different time points. RT–qPCR was performed from total RNA, and changes in mRNA expression were calculated and are depicted relative to the 0‐min time point (mean levels ± SEM, normalized to GUSB ; n = 2). Summary of microarray gene expression analysis performed in HeLa cells ± IL‐1α stimulation for 60 min on control (empty vector; n = 4) and three p65 enhancer‐deletion lines (Δp65 eIL8 / ‐ CXCL2 / ‐ IL8+CXCL2 ; n = 2). Differentially expressed genes were identified based on a moderated t ‐test ( P‐ value

    Article Snippet: Primary antibodies against the following proteins or peptides were used: anti‐β‐actin (Santa Cruz, #sc‐4778), anti‐CRISPR‐Cas9 (Abcam, #191468), anti‐CTCF (Millipore, #07‐729), anti‐FLAG (Sigma‐Aldrich, #F1804), anti‐H3 (Abcam, #ab1791), anti‐H3K4me1 (Abcam, #ab8895), anti‐H3K27ac (Diagenode, Pab‐174‐050), anti‐H3K27me3 (Millipore, #07‐449), anti‐H3K36ac (Diagenode, #C15410307), anti‐(PS32)‐IκBα (Cell Signaling, #2859), anti‐IκBα (Cell Signaling, #9242), anti‐P(T183/Y185)‐JNK (Cell Signaling, #9251), anti‐JNK (Santa Cruz, #sc‐571), anti‐P(S536)‐p65 (Cell Signaling, #3033), anti‐p65 (Santa Cruz, #sc‐372; #sc‐8008), anti‐P(T180/Y182)‐p38 (Zymed, #36‐8500), anti‐p38 (Cell Signaling, #9212), anti‐puromycin (3RH11; Kerafast, #EQ0001), anti‐P(S2)‐RNA Pol II (Abcam, #ab5095), anti‐P(S5)‐Pol II (Abcam, #ab5131), anti‐RNA‐Pol II (Millipore, #17‐620), anti‐tubulin (Santa Cruz, #sc‐8035), and normal rabbit IgG (Santa Cruz, #sc‐2027; Cell Signaling #2729).

    Techniques: Expressing, Mutagenesis, Plasmid Preparation, Quantitative RT-PCR, Microarray

    Heterologous activation of the IL8 enhancer triggers IL8 and CXCL1 expression A CRISPR activation (CRISPRa) strategy was applied to test enhancer functions individually. This approach involves a “dead” Cas9 ( blue ) and VP64 ( green ) fusion protein that recruits the NF‐κB ( orange ) and HSF1 ( red ) transactivation domains via MS2 recognition of two stem loops in the sgRNA scaffold ( magenta ). These complexes were targeted to the IL8 or CXCL2 enhancers and promoters (highlights) via different sgRNA pools in HeLa. The position of individual sgRNAs used for CRISPRa is shown in more detail in Fig EV1 . Left bar graphs: Wild‐type HeLa cells were transiently transfected with different combinations of plasmids encoding the “dead” Cas9‐VP64 fusion protein, the MS2‐p65‐HSF1 fusion protein, and empty sgRNA vector or versions containing sgRNAs targeting the IL8 enhancer or promoter. Twenty‐four hours post‐transfection, cells were lysed and total RNA was analyzed for expression changes of the indicated genes compared to samples carrying dCas9‐VP64 and MS2‐p65‐HSF1 fusions, but no sgRNAs. Right bar graphs: The same experiments were performed using sgRNAs targeting the CXCL2 enhancer and promoter. Data information: All data are from four independent transfections. Shown are mean values ± SEM. P ‐values are derived from unpaired t ‐tests comparing every condition against cells expressing all transactivators but lacking sgRNAs (first lane in each graph). Only significant differences are marked by asterisks.

    Journal: The EMBO Journal

    Article Title: Distinct IL‐1α‐responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

    doi: 10.15252/embj.2019101533

    Figure Lengend Snippet: Heterologous activation of the IL8 enhancer triggers IL8 and CXCL1 expression A CRISPR activation (CRISPRa) strategy was applied to test enhancer functions individually. This approach involves a “dead” Cas9 ( blue ) and VP64 ( green ) fusion protein that recruits the NF‐κB ( orange ) and HSF1 ( red ) transactivation domains via MS2 recognition of two stem loops in the sgRNA scaffold ( magenta ). These complexes were targeted to the IL8 or CXCL2 enhancers and promoters (highlights) via different sgRNA pools in HeLa. The position of individual sgRNAs used for CRISPRa is shown in more detail in Fig EV1 . Left bar graphs: Wild‐type HeLa cells were transiently transfected with different combinations of plasmids encoding the “dead” Cas9‐VP64 fusion protein, the MS2‐p65‐HSF1 fusion protein, and empty sgRNA vector or versions containing sgRNAs targeting the IL8 enhancer or promoter. Twenty‐four hours post‐transfection, cells were lysed and total RNA was analyzed for expression changes of the indicated genes compared to samples carrying dCas9‐VP64 and MS2‐p65‐HSF1 fusions, but no sgRNAs. Right bar graphs: The same experiments were performed using sgRNAs targeting the CXCL2 enhancer and promoter. Data information: All data are from four independent transfections. Shown are mean values ± SEM. P ‐values are derived from unpaired t ‐tests comparing every condition against cells expressing all transactivators but lacking sgRNAs (first lane in each graph). Only significant differences are marked by asterisks.

    Article Snippet: Primary antibodies against the following proteins or peptides were used: anti‐β‐actin (Santa Cruz, #sc‐4778), anti‐CRISPR‐Cas9 (Abcam, #191468), anti‐CTCF (Millipore, #07‐729), anti‐FLAG (Sigma‐Aldrich, #F1804), anti‐H3 (Abcam, #ab1791), anti‐H3K4me1 (Abcam, #ab8895), anti‐H3K27ac (Diagenode, Pab‐174‐050), anti‐H3K27me3 (Millipore, #07‐449), anti‐H3K36ac (Diagenode, #C15410307), anti‐(PS32)‐IκBα (Cell Signaling, #2859), anti‐IκBα (Cell Signaling, #9242), anti‐P(T183/Y185)‐JNK (Cell Signaling, #9251), anti‐JNK (Santa Cruz, #sc‐571), anti‐P(S536)‐p65 (Cell Signaling, #3033), anti‐p65 (Santa Cruz, #sc‐372; #sc‐8008), anti‐P(T180/Y182)‐p38 (Zymed, #36‐8500), anti‐p38 (Cell Signaling, #9212), anti‐puromycin (3RH11; Kerafast, #EQ0001), anti‐P(S2)‐RNA Pol II (Abcam, #ab5095), anti‐P(S5)‐Pol II (Abcam, #ab5131), anti‐RNA‐Pol II (Millipore, #17‐620), anti‐tubulin (Santa Cruz, #sc‐8035), and normal rabbit IgG (Santa Cruz, #sc‐2027; Cell Signaling #2729).

    Techniques: Activation Assay, Expressing, CRISPR, Transfection, Plasmid Preparation, Derivative Assay

    Intronic RNA FISH reveals reduced concomitant biallelic and colocalizing chemokine expression in enhancer‐mutant HeLa Representative triple RNA FISH images from HeLa cells ± IL‐1α stimulation for 60 min. Mature mRNAs ( IL8 , β‐actin ; red ) and intronic RNAs ( CXCL2 , purple ; IL8, green ) are detected against nuclei stained with Hoechst 33342 ( blue ). Typical foci marking individually labeled IL8 / CXCL2 transcription sites or merged signals indicating co‐transcription and spatial proximity are enlarged ( inset ). Scale bar: 10 μm. Quantification of RNA FISH signals from parental (wt), control (vector), p65‐deletion (Δp65 eIL8 and Δp65 eCXCL2 ), or p65‐knockout (Δ RELA ) HeLa lines ± IL‐1α stimulation for 60 min. Negative controls (neg ctrl) indicate samples from IL‐1α‐stimulated control cells in which RNA FISH was performed using pre‐amplifier, amplifier, and label probe mixes, but omitting the specific probe sets for IL8 or CXCL2 . These samples were used to define unspecific signals. Data from three independent experiments are pooled and plotted. The box plots show the distributions of FISH signals. Boundaries of the box indicate the 25 th /75 th percentiles, black lines mark medians, and colored lines mark means, respectively. Whiskers (error bars) indicate the 10 th /90 th percentiles, and circles mark all remaining outliers. The data from panel (B) were used to separately quantify the fraction of cells with mono‐ or biallelic IL8 or CXCL2 intronic RNA expression ( purple, green, blue colors ), as well as the extent of colocalizing (overlapping) intronic RNA FISH signals in individual cells, indicating simultaneously activated transcription sites on the same allele ( yellow colors ). The total numbers of cells analyzed are shown above each bar. Data are depicted relative to the total number of analyzed cells. * P

    Journal: The EMBO Journal

    Article Title: Distinct IL‐1α‐responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

    doi: 10.15252/embj.2019101533

    Figure Lengend Snippet: Intronic RNA FISH reveals reduced concomitant biallelic and colocalizing chemokine expression in enhancer‐mutant HeLa Representative triple RNA FISH images from HeLa cells ± IL‐1α stimulation for 60 min. Mature mRNAs ( IL8 , β‐actin ; red ) and intronic RNAs ( CXCL2 , purple ; IL8, green ) are detected against nuclei stained with Hoechst 33342 ( blue ). Typical foci marking individually labeled IL8 / CXCL2 transcription sites or merged signals indicating co‐transcription and spatial proximity are enlarged ( inset ). Scale bar: 10 μm. Quantification of RNA FISH signals from parental (wt), control (vector), p65‐deletion (Δp65 eIL8 and Δp65 eCXCL2 ), or p65‐knockout (Δ RELA ) HeLa lines ± IL‐1α stimulation for 60 min. Negative controls (neg ctrl) indicate samples from IL‐1α‐stimulated control cells in which RNA FISH was performed using pre‐amplifier, amplifier, and label probe mixes, but omitting the specific probe sets for IL8 or CXCL2 . These samples were used to define unspecific signals. Data from three independent experiments are pooled and plotted. The box plots show the distributions of FISH signals. Boundaries of the box indicate the 25 th /75 th percentiles, black lines mark medians, and colored lines mark means, respectively. Whiskers (error bars) indicate the 10 th /90 th percentiles, and circles mark all remaining outliers. The data from panel (B) were used to separately quantify the fraction of cells with mono‐ or biallelic IL8 or CXCL2 intronic RNA expression ( purple, green, blue colors ), as well as the extent of colocalizing (overlapping) intronic RNA FISH signals in individual cells, indicating simultaneously activated transcription sites on the same allele ( yellow colors ). The total numbers of cells analyzed are shown above each bar. Data are depicted relative to the total number of analyzed cells. * P

    Article Snippet: Primary antibodies against the following proteins or peptides were used: anti‐β‐actin (Santa Cruz, #sc‐4778), anti‐CRISPR‐Cas9 (Abcam, #191468), anti‐CTCF (Millipore, #07‐729), anti‐FLAG (Sigma‐Aldrich, #F1804), anti‐H3 (Abcam, #ab1791), anti‐H3K4me1 (Abcam, #ab8895), anti‐H3K27ac (Diagenode, Pab‐174‐050), anti‐H3K27me3 (Millipore, #07‐449), anti‐H3K36ac (Diagenode, #C15410307), anti‐(PS32)‐IκBα (Cell Signaling, #2859), anti‐IκBα (Cell Signaling, #9242), anti‐P(T183/Y185)‐JNK (Cell Signaling, #9251), anti‐JNK (Santa Cruz, #sc‐571), anti‐P(S536)‐p65 (Cell Signaling, #3033), anti‐p65 (Santa Cruz, #sc‐372; #sc‐8008), anti‐P(T180/Y182)‐p38 (Zymed, #36‐8500), anti‐p38 (Cell Signaling, #9212), anti‐puromycin (3RH11; Kerafast, #EQ0001), anti‐P(S2)‐RNA Pol II (Abcam, #ab5095), anti‐P(S5)‐Pol II (Abcam, #ab5131), anti‐RNA‐Pol II (Millipore, #17‐620), anti‐tubulin (Santa Cruz, #sc‐8035), and normal rabbit IgG (Santa Cruz, #sc‐2027; Cell Signaling #2729).

    Techniques: Fluorescence In Situ Hybridization, Expressing, Mutagenesis, Staining, Labeling, Plasmid Preparation, Knock-Out, RNA Expression

    IKK is required for glutamate/CHPG induction of p65 phosphorylation. (a) IKKα was immunoprecipitated from untreated (Untr.) and TNF- or CHPG-treated U87-MG cells and incubated with GST-IκBα or GST-p65 and [γ- 32 P]ATP. Proteins were resolved by SDS-PAGE and visualized by autoradiography. (b) U87-MG cells were pretreated with DMSO or compound A (Cmpd A) for 1 h, and cells were subsequently treated with glutamate (Glu; 400 μM), CHPG (200 μM), or TNF-α (10 ng/ml) for 10 min. Whole-cell lysates were isolated, and Western blot analysis was performed using the indicated antibodies. untr., untreated. (c) H4 cells were transfected with siRNA targeting IKKα. Twenty-four hours after transfection, the cells were serum starved for 14 to 16 h and subsequently treated with CHPG or TNF-α for 10 min. Whole-cell lysates were isolated, and Western blot analysis was performed with the indicated antibodies. (d) H4 cells were transfected with siRNA targeting IKKβ and analyzed as described for panel b.

    Journal: Molecular and Cellular Biology

    Article Title: Essential Role for Epidermal Growth Factor Receptor in Glutamate Receptor Signaling to NF-κB

    doi: 10.1128/MCB.00578-08

    Figure Lengend Snippet: IKK is required for glutamate/CHPG induction of p65 phosphorylation. (a) IKKα was immunoprecipitated from untreated (Untr.) and TNF- or CHPG-treated U87-MG cells and incubated with GST-IκBα or GST-p65 and [γ- 32 P]ATP. Proteins were resolved by SDS-PAGE and visualized by autoradiography. (b) U87-MG cells were pretreated with DMSO or compound A (Cmpd A) for 1 h, and cells were subsequently treated with glutamate (Glu; 400 μM), CHPG (200 μM), or TNF-α (10 ng/ml) for 10 min. Whole-cell lysates were isolated, and Western blot analysis was performed using the indicated antibodies. untr., untreated. (c) H4 cells were transfected with siRNA targeting IKKα. Twenty-four hours after transfection, the cells were serum starved for 14 to 16 h and subsequently treated with CHPG or TNF-α for 10 min. Whole-cell lysates were isolated, and Western blot analysis was performed with the indicated antibodies. (d) H4 cells were transfected with siRNA targeting IKKβ and analyzed as described for panel b.

    Article Snippet: For supershifts, 1 μl of anti-p65 antibody (SC-109; Santa Cruz) or 2 μl of anti-p50 antibody (SC-7178; Santa Cruz) was added to the binding reaction.

    Techniques: Immunoprecipitation, Incubation, SDS Page, Autoradiography, Isolation, Western Blot, Transfection

    Glutamate and mGluR5 stimulation induce the phosphorylation of p65 and IKK. Western blot analysis was performed on whole-cell lysates from H4 cells (a) and mouse primary astrocytes (b) treated with TNF-α, EGF, glutamate, or CHPG for 10 min using the indicated antibodies. Similar results were seen with U87-MG cells (data not shown). untr., untreated. (c) H4 cells were transfected with siRNA targeting mGluR5 and subsequently treated with glutamate (Glu) for 15 min. Western blot analysis was performed on whole-cell lysates using the indicated antibodies. unt., untreated.

    Journal: Molecular and Cellular Biology

    Article Title: Essential Role for Epidermal Growth Factor Receptor in Glutamate Receptor Signaling to NF-κB

    doi: 10.1128/MCB.00578-08

    Figure Lengend Snippet: Glutamate and mGluR5 stimulation induce the phosphorylation of p65 and IKK. Western blot analysis was performed on whole-cell lysates from H4 cells (a) and mouse primary astrocytes (b) treated with TNF-α, EGF, glutamate, or CHPG for 10 min using the indicated antibodies. Similar results were seen with U87-MG cells (data not shown). untr., untreated. (c) H4 cells were transfected with siRNA targeting mGluR5 and subsequently treated with glutamate (Glu) for 15 min. Western blot analysis was performed on whole-cell lysates using the indicated antibodies. unt., untreated.

    Article Snippet: For supershifts, 1 μl of anti-p65 antibody (SC-109; Santa Cruz) or 2 μl of anti-p50 antibody (SC-7178; Santa Cruz) was added to the binding reaction.

    Techniques: Western Blot, Transfection

    EGFR inhibition blocks cell proliferation and calcium signaling. (a) H4 cells were pretreated with DMSO or lapatinib (1 μM) for 1 h and subsequently treated with CHPG at the indicated doses for 10 min. Calcium mobilization was assayed using the Fluo4NW kit (Invitrogen). Error bars indicate the standard deviations ( n = 3). (b) H4 cells were pretreated with DMSO, lapatinib, gefitinib, or compound A (Cmpd A) (1 μM each) for 1 h and subsequently treated with glutamate (Glu; 400 μM), CHPG (200 μM), or EGF (100 ng/ml) for 24 h. Cell metabolic activity was measured using a colorimetric assay (Promega). Error bars indicate the standard deviations ( n = 4). (c) Model for glutamate signaling to NF-κB. Glutamate binding to mGluR5 triggers association with and transactivation of the EGFR leading to calcium mobilization, calcium-dependent IKKα/β phosphorylation, and dissociation of the p65-IκBα complexes. Glutamate also leads to p65 phosphorylation and enhanced p65 transcriptional activation. Circled P, Phosphorylation sites.

    Journal: Molecular and Cellular Biology

    Article Title: Essential Role for Epidermal Growth Factor Receptor in Glutamate Receptor Signaling to NF-κB

    doi: 10.1128/MCB.00578-08

    Figure Lengend Snippet: EGFR inhibition blocks cell proliferation and calcium signaling. (a) H4 cells were pretreated with DMSO or lapatinib (1 μM) for 1 h and subsequently treated with CHPG at the indicated doses for 10 min. Calcium mobilization was assayed using the Fluo4NW kit (Invitrogen). Error bars indicate the standard deviations ( n = 3). (b) H4 cells were pretreated with DMSO, lapatinib, gefitinib, or compound A (Cmpd A) (1 μM each) for 1 h and subsequently treated with glutamate (Glu; 400 μM), CHPG (200 μM), or EGF (100 ng/ml) for 24 h. Cell metabolic activity was measured using a colorimetric assay (Promega). Error bars indicate the standard deviations ( n = 4). (c) Model for glutamate signaling to NF-κB. Glutamate binding to mGluR5 triggers association with and transactivation of the EGFR leading to calcium mobilization, calcium-dependent IKKα/β phosphorylation, and dissociation of the p65-IκBα complexes. Glutamate also leads to p65 phosphorylation and enhanced p65 transcriptional activation. Circled P, Phosphorylation sites.

    Article Snippet: For supershifts, 1 μl of anti-p65 antibody (SC-109; Santa Cruz) or 2 μl of anti-p50 antibody (SC-7178; Santa Cruz) was added to the binding reaction.

    Techniques: Inhibition, Activity Assay, Colorimetric Assay, Binding Assay, Activation Assay

    Glutamate regulates NF-κB DNA binding and transcriptional activity. (a) EMSAs were performed using nuclear extracts from glutamate or TNF-treated H4 cells and radiolabeled oligonucleotides corresponding to an NF-κB consensus site from the κ light-chain enhancer (Promega) or the human EAAT2 promoter. Supershift assays were performed with the indicated antibodies. Arrows indicate p65-containing complexes. *, supershifted complexes. (b) ChIP assays were performed using IgG or p65 antibody in U87-MG cells that were untreated or treated with glutamate at the indicated time points. PCR analysis was performed using primers spanning the −583 NF-κB site of the EAAT2 promoter (see Materials and Methods). (c) A wild-type (WT) or NF-κB mutant (Mut) EAAT2-luciferase (luc) reporter was transfected into H4 cells and treated with glutamate or EGF at the indicated doses for 14 to 16 h. Whole-cell lysates were prepared and monitored for luciferase activity. The relative luciferase values represent units of luciferase normalized to renilla. The results from a representative experiment are shown. Error bars represent standard deviations ( n = 3). Glu, glutamate.

    Journal: Molecular and Cellular Biology

    Article Title: Essential Role for Epidermal Growth Factor Receptor in Glutamate Receptor Signaling to NF-κB

    doi: 10.1128/MCB.00578-08

    Figure Lengend Snippet: Glutamate regulates NF-κB DNA binding and transcriptional activity. (a) EMSAs were performed using nuclear extracts from glutamate or TNF-treated H4 cells and radiolabeled oligonucleotides corresponding to an NF-κB consensus site from the κ light-chain enhancer (Promega) or the human EAAT2 promoter. Supershift assays were performed with the indicated antibodies. Arrows indicate p65-containing complexes. *, supershifted complexes. (b) ChIP assays were performed using IgG or p65 antibody in U87-MG cells that were untreated or treated with glutamate at the indicated time points. PCR analysis was performed using primers spanning the −583 NF-κB site of the EAAT2 promoter (see Materials and Methods). (c) A wild-type (WT) or NF-κB mutant (Mut) EAAT2-luciferase (luc) reporter was transfected into H4 cells and treated with glutamate or EGF at the indicated doses for 14 to 16 h. Whole-cell lysates were prepared and monitored for luciferase activity. The relative luciferase values represent units of luciferase normalized to renilla. The results from a representative experiment are shown. Error bars represent standard deviations ( n = 3). Glu, glutamate.

    Article Snippet: For supershifts, 1 μl of anti-p65 antibody (SC-109; Santa Cruz) or 2 μl of anti-p50 antibody (SC-7178; Santa Cruz) was added to the binding reaction.

    Techniques: Binding Assay, Activity Assay, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Mutagenesis, Luciferase, Transfection

    Glutamate/mGluR5 attenuates p65-IκBα interaction in the absence of IκBα phosphorylation/degradation. (a) Whole-cell lysates were isolated from H4 cells treated with TNF-α (10 ng/ml), EGF (100 ng/ml), or glutamate (400 μM) for 30 min. Western blot analysis was performed on 10 to 30 μg protein using antibodies to phospho-IκBα and total IκBα. (b) Untreated U87-MG cells or cells treated with 1 μM of MG-132 were stimulated with glutamate (400 μM), EGF (100 ng/ml), or TNF-α (20 ng/ml) for 15 min, and whole-cell lysates were harvested for Western blot analysis to examine phospho-IκBα-Ser32/36 and total IκBα to verify the efficacy of proteosome inhibition. Western blot analysis was also performed using antibodies to phospho-p65-Ser536 and total p65. Similarly treated cells were used to isolate nuclear fractions for EMSA using the κ light-chain enhancer NF-κB probe. (c) Antibodies to IgG or p65 (5 μg each) were used to immunoprecipitate endogenous proteins from 250 μg of U87-MG whole-cell lysates that were untreated (U) or treated with TNF-α (T; 20 ng/ml) or glutamate (G; 400 μM) for 30 min. Western blot analysis was performed on immunoprecipitates using antibodies to IκB, p50 (SC-7178; Santa Cruz) or p65 (SC-109; Santa Cruz). The pixel densities of IκBα and p65 Western blots were quantitated using NIH Scion software. Graphs represent the pixel density for IκBα normalized to p65 for each experimental condition. Unt, untreated; Glu, glutamate; IP, immunoprecipitate.

    Journal: Molecular and Cellular Biology

    Article Title: Essential Role for Epidermal Growth Factor Receptor in Glutamate Receptor Signaling to NF-κB

    doi: 10.1128/MCB.00578-08

    Figure Lengend Snippet: Glutamate/mGluR5 attenuates p65-IκBα interaction in the absence of IκBα phosphorylation/degradation. (a) Whole-cell lysates were isolated from H4 cells treated with TNF-α (10 ng/ml), EGF (100 ng/ml), or glutamate (400 μM) for 30 min. Western blot analysis was performed on 10 to 30 μg protein using antibodies to phospho-IκBα and total IκBα. (b) Untreated U87-MG cells or cells treated with 1 μM of MG-132 were stimulated with glutamate (400 μM), EGF (100 ng/ml), or TNF-α (20 ng/ml) for 15 min, and whole-cell lysates were harvested for Western blot analysis to examine phospho-IκBα-Ser32/36 and total IκBα to verify the efficacy of proteosome inhibition. Western blot analysis was also performed using antibodies to phospho-p65-Ser536 and total p65. Similarly treated cells were used to isolate nuclear fractions for EMSA using the κ light-chain enhancer NF-κB probe. (c) Antibodies to IgG or p65 (5 μg each) were used to immunoprecipitate endogenous proteins from 250 μg of U87-MG whole-cell lysates that were untreated (U) or treated with TNF-α (T; 20 ng/ml) or glutamate (G; 400 μM) for 30 min. Western blot analysis was performed on immunoprecipitates using antibodies to IκB, p50 (SC-7178; Santa Cruz) or p65 (SC-109; Santa Cruz). The pixel densities of IκBα and p65 Western blots were quantitated using NIH Scion software. Graphs represent the pixel density for IκBα normalized to p65 for each experimental condition. Unt, untreated; Glu, glutamate; IP, immunoprecipitate.

    Article Snippet: For supershifts, 1 μl of anti-p65 antibody (SC-109; Santa Cruz) or 2 μl of anti-p50 antibody (SC-7178; Santa Cruz) was added to the binding reaction.

    Techniques: Isolation, Western Blot, Inhibition, Software

    Glutamate induces p65 nuclear accumulation and transactivation. Nuclear and cytoplasmic fractions were isolated from U87-MG cells treated with glutamate (Glu; 400 μM), EGF (100 ng/ml), or TNF-α (20 ng/ml) (a) or CHPG (200 μM) (b) at the indicated time points. Western blot analysis was performed using the indicated antibodies. β-Tubulin and lamin A/C were used as controls for loading and subcellular fractionation. Unt., untreated. (c) Glutamate increases p65 transcriptional activation. H4 cells were transiently transfected with a Gal4-luciferase reporter (Gal4-luc) and either Gal4-Elk1(TAD) or Gal4-p65(TAD) fusion proteins. Cells were serum starved overnight and stimulated with glutamate (Glu; 400 μM), EGF (100 ng/ml), or TNF-α (20 ng/ml) for 14 to 16 h and monitored for luciferase and renilla activity. Relative luciferase values represent units of luciferase normalized to renilla. Error bars represent standard deviations ( n = 3). unt, untreated.

    Journal: Molecular and Cellular Biology

    Article Title: Essential Role for Epidermal Growth Factor Receptor in Glutamate Receptor Signaling to NF-κB

    doi: 10.1128/MCB.00578-08

    Figure Lengend Snippet: Glutamate induces p65 nuclear accumulation and transactivation. Nuclear and cytoplasmic fractions were isolated from U87-MG cells treated with glutamate (Glu; 400 μM), EGF (100 ng/ml), or TNF-α (20 ng/ml) (a) or CHPG (200 μM) (b) at the indicated time points. Western blot analysis was performed using the indicated antibodies. β-Tubulin and lamin A/C were used as controls for loading and subcellular fractionation. Unt., untreated. (c) Glutamate increases p65 transcriptional activation. H4 cells were transiently transfected with a Gal4-luciferase reporter (Gal4-luc) and either Gal4-Elk1(TAD) or Gal4-p65(TAD) fusion proteins. Cells were serum starved overnight and stimulated with glutamate (Glu; 400 μM), EGF (100 ng/ml), or TNF-α (20 ng/ml) for 14 to 16 h and monitored for luciferase and renilla activity. Relative luciferase values represent units of luciferase normalized to renilla. Error bars represent standard deviations ( n = 3). unt, untreated.

    Article Snippet: For supershifts, 1 μl of anti-p65 antibody (SC-109; Santa Cruz) or 2 μl of anti-p50 antibody (SC-7178; Santa Cruz) was added to the binding reaction.

    Techniques: Isolation, Western Blot, Fractionation, Activation Assay, Transfection, Luciferase, Activity Assay

    Intracellular calcium is required for CHPG-mediated p65 phosphorylation. (a) U87-MG cells were pretreated with DMSO, BAPTA-AM (10 μM), or TPEN (10 μM) for 1 h and subsequently treated with glutamate (Glu; 400 μM) or EGF (100 ng/ml) for 10 min. Whole-cell lysates were prepared for Western blot analysis with the indicated antibodies. unt., untreated. (b) H4 cells were pretreated with DMSO or BAPTA-AM (10 μM) for 1 h and subsequently treated with glutamate (Glu; 400 μM) or TNF-α (10 ng/ml) for 10 min. Whole-cell lysates were prepared for Western blot analysis with the indicated antibodies to examine phosphorylated and total IKKα/β. The pixel densities of the phospho-IKKα/β bands were determined using NIH Scion software and normalized to untreated DMSO (lane 1) or untreated BAPTA-AM (lane 5) to examine n -fold induction.

    Journal: Molecular and Cellular Biology

    Article Title: Essential Role for Epidermal Growth Factor Receptor in Glutamate Receptor Signaling to NF-κB

    doi: 10.1128/MCB.00578-08

    Figure Lengend Snippet: Intracellular calcium is required for CHPG-mediated p65 phosphorylation. (a) U87-MG cells were pretreated with DMSO, BAPTA-AM (10 μM), or TPEN (10 μM) for 1 h and subsequently treated with glutamate (Glu; 400 μM) or EGF (100 ng/ml) for 10 min. Whole-cell lysates were prepared for Western blot analysis with the indicated antibodies. unt., untreated. (b) H4 cells were pretreated with DMSO or BAPTA-AM (10 μM) for 1 h and subsequently treated with glutamate (Glu; 400 μM) or TNF-α (10 ng/ml) for 10 min. Whole-cell lysates were prepared for Western blot analysis with the indicated antibodies to examine phosphorylated and total IKKα/β. The pixel densities of the phospho-IKKα/β bands were determined using NIH Scion software and normalized to untreated DMSO (lane 1) or untreated BAPTA-AM (lane 5) to examine n -fold induction.

    Article Snippet: For supershifts, 1 μl of anti-p65 antibody (SC-109; Santa Cruz) or 2 μl of anti-p50 antibody (SC-7178; Santa Cruz) was added to the binding reaction.

    Techniques: Western Blot, Software

    Fargesin blocked the activation of NF-κB in vitro. ( A ) RAW264.7 cells were treated as described in the Methods and p-p65 localization was visualized under a fluorescence microscope. Scale bar corresponds to 200 μm. ( B ) RAW264.7 cells were treated as described in the Methods. NF-κB promoter-driven luciferase activity was determined using a dual luciferase assay system, and values were expressed as the fold induction of the control cells. Data were expressed as mean ± SD of quadruplicates of two independent experiments. *** p

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: Anti-Inflammatory Effects of Fargesin on Chemically Induced Inflammatory Bowel Disease in Mice

    doi: 10.3390/molecules23061380

    Figure Lengend Snippet: Fargesin blocked the activation of NF-κB in vitro. ( A ) RAW264.7 cells were treated as described in the Methods and p-p65 localization was visualized under a fluorescence microscope. Scale bar corresponds to 200 μm. ( B ) RAW264.7 cells were treated as described in the Methods. NF-κB promoter-driven luciferase activity was determined using a dual luciferase assay system, and values were expressed as the fold induction of the control cells. Data were expressed as mean ± SD of quadruplicates of two independent experiments. *** p

    Article Snippet: The following antibodies—NF-κB p65 (#8242), phospho-p65 (#3033), IκBα (#4812), phospho-IκBα (#2859), and β-actin (#4970)—were obtained from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Activation Assay, In Vitro, Fluorescence, Microscopy, Luciferase, Activity Assay

    Fargesin inhibited the activation of NF-κB in vivo. ( A ) Protein levels were determined by immunoblotting using p-p65 (1:1000), p-IκBα (1:1000) and IκBα (1:1000) antibodies. One representative experiment is shown. Data were expressed as the mean ± SD of two independent experiments with samples in triplicate. Quantification of the protein expression was performed by densitometric analysis of the blots. ( B ) Representative images of p-p65 immunostaining in colon tissue. Scale bar corresponds to 100 μm ** p

    Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

    Article Title: Anti-Inflammatory Effects of Fargesin on Chemically Induced Inflammatory Bowel Disease in Mice

    doi: 10.3390/molecules23061380

    Figure Lengend Snippet: Fargesin inhibited the activation of NF-κB in vivo. ( A ) Protein levels were determined by immunoblotting using p-p65 (1:1000), p-IκBα (1:1000) and IκBα (1:1000) antibodies. One representative experiment is shown. Data were expressed as the mean ± SD of two independent experiments with samples in triplicate. Quantification of the protein expression was performed by densitometric analysis of the blots. ( B ) Representative images of p-p65 immunostaining in colon tissue. Scale bar corresponds to 100 μm ** p

    Article Snippet: The following antibodies—NF-κB p65 (#8242), phospho-p65 (#3033), IκBα (#4812), phospho-IκBα (#2859), and β-actin (#4970)—were obtained from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Activation Assay, In Vivo, Expressing, Immunostaining

    Inhibitory effects of cecropin-TY1 on LPS-stimulated inflammatory response signal pathway in peritoneal macrophages. a – b Effects of cecropin-TY1 on ( a ) MAPKs and ( b ) NF-κB activation in peritoneal macrophages after 100 ng/mL LPS stimulation. The photograph is a representative one of three independent experiments. Peritoneal macrophages were stimulated with or without LPS (100 ng/mL), then different concentrations of cecropin-TY1 (cec-TY1, 5, 10, 20 μg/mL) were added immediately and incubated for 30 min, and macrophages were lysed for western blot analysis. ( c – i Ratio of ( c ) P-ERK1 (42 kDa), ( d ) P-ERK2 (44 kDa), ( e ) P-JNK1 (46 kDa), ( f ) P-JNK2 (54 kDa), ( g ) P-p38, ( h ) P-IκBα and ( i ) P-p65 to β-actin. Band densities were analyzed using Quantity One software (Bio-Rad, Richmond, CA USA). Data were presented as mean ± SEM. * P

    Journal: Parasites & Vectors

    Article Title: A potent anti-inflammatory peptide from the salivary glands of horsefly

    doi: 10.1186/s13071-015-1149-y

    Figure Lengend Snippet: Inhibitory effects of cecropin-TY1 on LPS-stimulated inflammatory response signal pathway in peritoneal macrophages. a – b Effects of cecropin-TY1 on ( a ) MAPKs and ( b ) NF-κB activation in peritoneal macrophages after 100 ng/mL LPS stimulation. The photograph is a representative one of three independent experiments. Peritoneal macrophages were stimulated with or without LPS (100 ng/mL), then different concentrations of cecropin-TY1 (cec-TY1, 5, 10, 20 μg/mL) were added immediately and incubated for 30 min, and macrophages were lysed for western blot analysis. ( c – i Ratio of ( c ) P-ERK1 (42 kDa), ( d ) P-ERK2 (44 kDa), ( e ) P-JNK1 (46 kDa), ( f ) P-JNK2 (54 kDa), ( g ) P-p38, ( h ) P-IκBα and ( i ) P-p65 to β-actin. Band densities were analyzed using Quantity One software (Bio-Rad, Richmond, CA USA). Data were presented as mean ± SEM. * P

    Article Snippet: The membrane was then incubated with primary antibodies against ERK, phospho-ERK, p38, phospho-p38, JNK, phospho-JNK, phospho-IκBα, NF-κB p65 and phospho-NF-κB p65 (1:2000, Cell Signaling Technology, USA) and GAPDH/β-actin (1:5000, Santa Cruz Biotechnology, USA) overnight at 4 °C, respectively.

    Techniques: Activation Assay, Capillary Electrochromatography, Incubation, Western Blot, Software

    Quantification of IL‐1α‐mediated enhancer modifications and p65 NF‐κB binding in the human IL8 and CXCL2 chemokine loci Published ChIP‐seq data from KB cells (Jurida et al , 2015 ; GSE64224 and GSE52470) were used to annotate active enhancers and p65 NF‐κB recruitment in untreated cells compared to cells stimulated with IL‐1α for 60 min ± TAKi. The browser views show ChIP‐seq profiles for all conditions of the IL8 and CXCL2 chemokine loci. Gray areas highlight four chromatin regions with IL‐1α‐inducible H3K27 acetylation and p65 binding. As indicated by black horizontal bars, these chromatin regions were provisionally designated by us as “class II enhancers” (Jurida et al , 2015 ) to distinguish them from the entire repertoire of all active (i.e., H3K4me1‐ and H3K27ac‐positive) enhancers. Vertical bars indicate predicted NF‐κB motifs in the DNA sequence and the positions of all sgRNAs used in this study for CRISPRa (see Fig 6 ) or for enhancer or promoter deletions (see Figs 3 and EV4). Bar graphs show cumulative read counts across the four IL‐1α‐regulated and TAKi‐sensitive class II enhancers. The two flanking enhancers with strongest p65 binding (2 and 4) are the ones investigated in detail in this study.

    Journal: The EMBO Journal

    Article Title: Distinct IL‐1α‐responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

    doi: 10.15252/embj.2019101533

    Figure Lengend Snippet: Quantification of IL‐1α‐mediated enhancer modifications and p65 NF‐κB binding in the human IL8 and CXCL2 chemokine loci Published ChIP‐seq data from KB cells (Jurida et al , 2015 ; GSE64224 and GSE52470) were used to annotate active enhancers and p65 NF‐κB recruitment in untreated cells compared to cells stimulated with IL‐1α for 60 min ± TAKi. The browser views show ChIP‐seq profiles for all conditions of the IL8 and CXCL2 chemokine loci. Gray areas highlight four chromatin regions with IL‐1α‐inducible H3K27 acetylation and p65 binding. As indicated by black horizontal bars, these chromatin regions were provisionally designated by us as “class II enhancers” (Jurida et al , 2015 ) to distinguish them from the entire repertoire of all active (i.e., H3K4me1‐ and H3K27ac‐positive) enhancers. Vertical bars indicate predicted NF‐κB motifs in the DNA sequence and the positions of all sgRNAs used in this study for CRISPRa (see Fig 6 ) or for enhancer or promoter deletions (see Figs 3 and EV4). Bar graphs show cumulative read counts across the four IL‐1α‐regulated and TAKi‐sensitive class II enhancers. The two flanking enhancers with strongest p65 binding (2 and 4) are the ones investigated in detail in this study.

    Article Snippet: For CHIP, the following antibodies were used: anti‐histone H3 (2 μg; Abcam; ab1791), anti‐NF‐κB p65 (3 μg; Santa Cruz; sc‐372), anti‐phospho‐Pol II (S5) (1.35 μg; Abcam; ab5131), H3K27ac (2 μg; Diagenode, pAb‐174‐050), H3K4me1 (2 μg; Abcam, ab8895), IgG (2 μg; Cell Signaling; 2729), and CTCF (4 μl; Millipore, 07‐729).

    Techniques: Binding Assay, Chromatin Immunoprecipitation, Sequencing

    Spatial chromatin interactions in the IL8 locus are rewired by deleting p65‐binding cis ‐elements within enhancers i4C profiles in the 1 Mbp around the IL8 locus on chromosome 4 ( ideogram ) from control (empty vector) and enhancer‐mutant (Δp65 eIL8 and Δp65 eCXCL2 ) HeLa lines ± IL‐1α stimulation for 60 min, generated using the IL8 promoter ( pink highlight ) or enhancer ( blue highlight ) as a viewpoint. For the IL8 promoter, the average of two biological replicates is shown, while for the IL8 enhancer, data from one replicate are shown. Below each profile, significantly strong ( brown ), medium ( red ), or weaker interactions ( orange ) called via foursig are indicated. All profiles are shown aligned to gene models ( blue ) and to CTCF, H3K4me1, H3K4me3, H3K27ac, and RNA polymerase II ENCODE HeLa‐S3 ChIP‐seq profiles. The breadth of TADs in the locus is indicated above. Meta‐plots showing coverage of H3K27ac ChIP‐seq signal at i4C fragments ± 1 kbp contacted by the IL8 promoter or enhancer in control cells (empty vector) in the presence ( magenta ) or absence ( gray ) of IL‐1α stimulation for 60 min, and in enhancer‐mutant cells (Δp65 eIL8 , blue ; Δp65 eCXCL2 , green ) after IL‐1α stimulation. Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Distinct IL‐1α‐responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

    doi: 10.15252/embj.2019101533

    Figure Lengend Snippet: Spatial chromatin interactions in the IL8 locus are rewired by deleting p65‐binding cis ‐elements within enhancers i4C profiles in the 1 Mbp around the IL8 locus on chromosome 4 ( ideogram ) from control (empty vector) and enhancer‐mutant (Δp65 eIL8 and Δp65 eCXCL2 ) HeLa lines ± IL‐1α stimulation for 60 min, generated using the IL8 promoter ( pink highlight ) or enhancer ( blue highlight ) as a viewpoint. For the IL8 promoter, the average of two biological replicates is shown, while for the IL8 enhancer, data from one replicate are shown. Below each profile, significantly strong ( brown ), medium ( red ), or weaker interactions ( orange ) called via foursig are indicated. All profiles are shown aligned to gene models ( blue ) and to CTCF, H3K4me1, H3K4me3, H3K27ac, and RNA polymerase II ENCODE HeLa‐S3 ChIP‐seq profiles. The breadth of TADs in the locus is indicated above. Meta‐plots showing coverage of H3K27ac ChIP‐seq signal at i4C fragments ± 1 kbp contacted by the IL8 promoter or enhancer in control cells (empty vector) in the presence ( magenta ) or absence ( gray ) of IL‐1α stimulation for 60 min, and in enhancer‐mutant cells (Δp65 eIL8 , blue ; Δp65 eCXCL2 , green ) after IL‐1α stimulation. Source data are available online for this figure.

    Article Snippet: For CHIP, the following antibodies were used: anti‐histone H3 (2 μg; Abcam; ab1791), anti‐NF‐κB p65 (3 μg; Santa Cruz; sc‐372), anti‐phospho‐Pol II (S5) (1.35 μg; Abcam; ab5131), H3K27ac (2 μg; Diagenode, pAb‐174‐050), H3K4me1 (2 μg; Abcam, ab8895), IgG (2 μg; Cell Signaling; 2729), and CTCF (4 μl; Millipore, 07‐729).

    Techniques: Binding Assay, Plasmid Preparation, Mutagenesis, Generated, Chromatin Immunoprecipitation

    Deletion of the p65‐binding site from the IL8 promoter only affects IL8 expression Genome browser view of the IL8 chemokine locus on human chromosome 4 showing H3K4me1, H3K4me3, H3K27ac, and RNA polymerase II ENCODE ChIP‐seq profiles from HeLa‐S3 cells relative to the IL8 gene model ( blue ). The location of the deleted NF‐κB binding site in the IL8 promoter is indicated ( orange ), and Sanger sequencing of PCR amplicons generated from genomic DNA of the Δp65 pIL8 cell line shows removal of 57 bp. Immunoblot analysis of extracts from parental HeLa cells (wt), vector controls, or Δp65 pIL8 cells was compared to cells stably expressing a sgRNA targeting the RELA (Δ RELA ) gene which encodes for the p65 NF‐κB subunit in the presence (+) or absence (−) of IL‐1α stimulation for 30 min or 60 min. Antibodies against p65 confirm the strong suppression of p65 in Δ RELA cells. Antibodies against P(S536)‐p65, P‐IκBα, and IκBα reveal normal IL‐1α‐mediated signaling in Δp65 pIL8 cells compared to control cells, while Δ RELA cells show reduced levels of the IL‐1α and the p65 target gene IκBα. β‐Actin levels provide a loading control. Cas9 antibodies reveal the levels of FLAG‐Cas9 present in the stable cell cultures. mRNA levels of seven IL‐1α‐responsive genes in parental (wt), control (empty vector), or promoter‐mutant (Δp65 pIL8 ) HeLa lines were assessed by RT–qPCR (mean levels ± SEM, n = 4) at 60 min after IL‐1α stimulation. Asterisks show significanc e of changes in basal and IL‐1α‐stimulated conditions compared to the corresponding vector control samples ( P

    Journal: The EMBO Journal

    Article Title: Distinct IL‐1α‐responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

    doi: 10.15252/embj.2019101533

    Figure Lengend Snippet: Deletion of the p65‐binding site from the IL8 promoter only affects IL8 expression Genome browser view of the IL8 chemokine locus on human chromosome 4 showing H3K4me1, H3K4me3, H3K27ac, and RNA polymerase II ENCODE ChIP‐seq profiles from HeLa‐S3 cells relative to the IL8 gene model ( blue ). The location of the deleted NF‐κB binding site in the IL8 promoter is indicated ( orange ), and Sanger sequencing of PCR amplicons generated from genomic DNA of the Δp65 pIL8 cell line shows removal of 57 bp. Immunoblot analysis of extracts from parental HeLa cells (wt), vector controls, or Δp65 pIL8 cells was compared to cells stably expressing a sgRNA targeting the RELA (Δ RELA ) gene which encodes for the p65 NF‐κB subunit in the presence (+) or absence (−) of IL‐1α stimulation for 30 min or 60 min. Antibodies against p65 confirm the strong suppression of p65 in Δ RELA cells. Antibodies against P(S536)‐p65, P‐IκBα, and IκBα reveal normal IL‐1α‐mediated signaling in Δp65 pIL8 cells compared to control cells, while Δ RELA cells show reduced levels of the IL‐1α and the p65 target gene IκBα. β‐Actin levels provide a loading control. Cas9 antibodies reveal the levels of FLAG‐Cas9 present in the stable cell cultures. mRNA levels of seven IL‐1α‐responsive genes in parental (wt), control (empty vector), or promoter‐mutant (Δp65 pIL8 ) HeLa lines were assessed by RT–qPCR (mean levels ± SEM, n = 4) at 60 min after IL‐1α stimulation. Asterisks show significanc e of changes in basal and IL‐1α‐stimulated conditions compared to the corresponding vector control samples ( P

    Article Snippet: For CHIP, the following antibodies were used: anti‐histone H3 (2 μg; Abcam; ab1791), anti‐NF‐κB p65 (3 μg; Santa Cruz; sc‐372), anti‐phospho‐Pol II (S5) (1.35 μg; Abcam; ab5131), H3K27ac (2 μg; Diagenode, pAb‐174‐050), H3K4me1 (2 μg; Abcam, ab8895), IgG (2 μg; Cell Signaling; 2729), and CTCF (4 μl; Millipore, 07‐729).

    Techniques: Binding Assay, Expressing, Chromatin Immunoprecipitation, Sequencing, Polymerase Chain Reaction, Generated, Plasmid Preparation, Stable Transfection, Mutagenesis, Quantitative RT-PCR

    The IL‐1α–TAK1 pathway regulates spatial chromatin interactions by the CXCL2 locus Cross‐linking‐free chromosome conformation capture (i4C) analysis was performed using chromatin from KB cells ± IL‐1α stimulation for 60 min in the presence or absence of a TAK inhibitor (TAKi). Shown are i4C profiles in the 1 Mbp around the CXCL2 locus on chromosome 4 ( ideogram ). Average read counts of two biological replicates are plotted, generated using the CXCL2 promoter ( blue highlight ) or enhancer ( pink highlight ) as a viewpoint. The region of the IL8 promoter/enhancer is also shown ( gray highlight ). Below each profile, significantly strong ( brown ), medium ( red ), or weaker interactions ( orange ) called via foursig software (Williams et al , 2014 ) are indicated. All profiles are shown aligned to gene models ( blue ) and CTCF ChIP‐seq, as well as to H3K27ac and H3K4me1 ChIP‐seq data from KB cells (GSE64224 + GSE52470) performed under the same conditions (Jurida et al , 2015 ). The breadth of topologically associating domains (TADs) in the locus is indicated above. Meta‐plots showing coverage of ATAC‐seq (this study) and H3K27ac, p65, and RNA polymerase II (RNAPII) ChIP‐seq signals (GSE64224 + GSE52470) at i4C fragments ± 1 kbp contacted by the CXCL2 promoter or enhancer in KB cells ± IL‐1α stimulation for 60 min in the presence or absence of a TAK inhibitor (TAKi). Source data are available online for this figure.

    Journal: The EMBO Journal

    Article Title: Distinct IL‐1α‐responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

    doi: 10.15252/embj.2019101533

    Figure Lengend Snippet: The IL‐1α–TAK1 pathway regulates spatial chromatin interactions by the CXCL2 locus Cross‐linking‐free chromosome conformation capture (i4C) analysis was performed using chromatin from KB cells ± IL‐1α stimulation for 60 min in the presence or absence of a TAK inhibitor (TAKi). Shown are i4C profiles in the 1 Mbp around the CXCL2 locus on chromosome 4 ( ideogram ). Average read counts of two biological replicates are plotted, generated using the CXCL2 promoter ( blue highlight ) or enhancer ( pink highlight ) as a viewpoint. The region of the IL8 promoter/enhancer is also shown ( gray highlight ). Below each profile, significantly strong ( brown ), medium ( red ), or weaker interactions ( orange ) called via foursig software (Williams et al , 2014 ) are indicated. All profiles are shown aligned to gene models ( blue ) and CTCF ChIP‐seq, as well as to H3K27ac and H3K4me1 ChIP‐seq data from KB cells (GSE64224 + GSE52470) performed under the same conditions (Jurida et al , 2015 ). The breadth of topologically associating domains (TADs) in the locus is indicated above. Meta‐plots showing coverage of ATAC‐seq (this study) and H3K27ac, p65, and RNA polymerase II (RNAPII) ChIP‐seq signals (GSE64224 + GSE52470) at i4C fragments ± 1 kbp contacted by the CXCL2 promoter or enhancer in KB cells ± IL‐1α stimulation for 60 min in the presence or absence of a TAK inhibitor (TAKi). Source data are available online for this figure.

    Article Snippet: For CHIP, the following antibodies were used: anti‐histone H3 (2 μg; Abcam; ab1791), anti‐NF‐κB p65 (3 μg; Santa Cruz; sc‐372), anti‐phospho‐Pol II (S5) (1.35 μg; Abcam; ab5131), H3K27ac (2 μg; Diagenode, pAb‐174‐050), H3K4me1 (2 μg; Abcam, ab8895), IgG (2 μg; Cell Signaling; 2729), and CTCF (4 μl; Millipore, 07‐729).

    Techniques: Generated, Software, Chromatin Immunoprecipitation

    Differential regulation of enhancers and promoters of IL‐1α target loci NF‐κB (p65), histone marks, and RNA polymerase II enrichment at the IL8 and CXCL2 promoter and enhancer in control (empty vector) or mutated (Δp65 eIL8 and Δp65 eCXCL2 ) HeLa lines were assessed by ChIP‐qPCR (mean enrichment over input ± SEM) at the indicated times after IL‐1α stimulation. IgG, H3K27ac, H3K4me1, H3, and p65 ChIP‐qPCR data are from three (vector, Δp65 eCXCL2 ) or four (Δp65 EIL8 ) independent experiments performed in duplicate; all others are from at least two independent experiments. *: significantly different to vector controls; P

    Journal: The EMBO Journal

    Article Title: Distinct IL‐1α‐responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

    doi: 10.15252/embj.2019101533

    Figure Lengend Snippet: Differential regulation of enhancers and promoters of IL‐1α target loci NF‐κB (p65), histone marks, and RNA polymerase II enrichment at the IL8 and CXCL2 promoter and enhancer in control (empty vector) or mutated (Δp65 eIL8 and Δp65 eCXCL2 ) HeLa lines were assessed by ChIP‐qPCR (mean enrichment over input ± SEM) at the indicated times after IL‐1α stimulation. IgG, H3K27ac, H3K4me1, H3, and p65 ChIP‐qPCR data are from three (vector, Δp65 eCXCL2 ) or four (Δp65 EIL8 ) independent experiments performed in duplicate; all others are from at least two independent experiments. *: significantly different to vector controls; P

    Article Snippet: For CHIP, the following antibodies were used: anti‐histone H3 (2 μg; Abcam; ab1791), anti‐NF‐κB p65 (3 μg; Santa Cruz; sc‐372), anti‐phospho‐Pol II (S5) (1.35 μg; Abcam; ab5131), H3K27ac (2 μg; Diagenode, pAb‐174‐050), H3K4me1 (2 μg; Abcam, ab8895), IgG (2 μg; Cell Signaling; 2729), and CTCF (4 μl; Millipore, 07‐729).

    Techniques: Plasmid Preparation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    Analyses of mRNA stabilities and the global IL‐1α gene expression response in enhancer‐mutant HeLa Empty vector controls or Δp65 eIL8 , Δp65 eCXCL2 , and Δp65 eIL8+eCXCL2 enhancer‐mutant HeLa lines were stimulated with IL‐1α for 60 min or were left untreated. Then, actinomycin D (5 μg/ml) was added to stop transcription and RNAs were immediately extracted (reference time point 0 min) or incubations were continued to monitor the decay of IL8 and CXCL2 mRNAs at different time points. RT–qPCR was performed from total RNA, and changes in mRNA expression were calculated and are depicted relative to the 0‐min time point (mean levels ± SEM, normalized to GUSB ; n = 2). Summary of microarray gene expression analysis performed in HeLa cells ± IL‐1α stimulation for 60 min on control (empty vector; n = 4) and three p65 enhancer‐deletion lines (Δp65 eIL8 / ‐ CXCL2 / ‐ IL8+CXCL2 ; n = 2). Differentially expressed genes were identified based on a moderated t ‐test ( P‐ value

    Journal: The EMBO Journal

    Article Title: Distinct IL‐1α‐responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

    doi: 10.15252/embj.2019101533

    Figure Lengend Snippet: Analyses of mRNA stabilities and the global IL‐1α gene expression response in enhancer‐mutant HeLa Empty vector controls or Δp65 eIL8 , Δp65 eCXCL2 , and Δp65 eIL8+eCXCL2 enhancer‐mutant HeLa lines were stimulated with IL‐1α for 60 min or were left untreated. Then, actinomycin D (5 μg/ml) was added to stop transcription and RNAs were immediately extracted (reference time point 0 min) or incubations were continued to monitor the decay of IL8 and CXCL2 mRNAs at different time points. RT–qPCR was performed from total RNA, and changes in mRNA expression were calculated and are depicted relative to the 0‐min time point (mean levels ± SEM, normalized to GUSB ; n = 2). Summary of microarray gene expression analysis performed in HeLa cells ± IL‐1α stimulation for 60 min on control (empty vector; n = 4) and three p65 enhancer‐deletion lines (Δp65 eIL8 / ‐ CXCL2 / ‐ IL8+CXCL2 ; n = 2). Differentially expressed genes were identified based on a moderated t ‐test ( P‐ value

    Article Snippet: For CHIP, the following antibodies were used: anti‐histone H3 (2 μg; Abcam; ab1791), anti‐NF‐κB p65 (3 μg; Santa Cruz; sc‐372), anti‐phospho‐Pol II (S5) (1.35 μg; Abcam; ab5131), H3K27ac (2 μg; Diagenode, pAb‐174‐050), H3K4me1 (2 μg; Abcam, ab8895), IgG (2 μg; Cell Signaling; 2729), and CTCF (4 μl; Millipore, 07‐729).

    Techniques: Expressing, Mutagenesis, Plasmid Preparation, Quantitative RT-PCR, Microarray

    Heterologous activation of the IL8 enhancer triggers IL8 and CXCL1 expression A CRISPR activation (CRISPRa) strategy was applied to test enhancer functions individually. This approach involves a “dead” Cas9 ( blue ) and VP64 ( green ) fusion protein that recruits the NF‐κB ( orange ) and HSF1 ( red ) transactivation domains via MS2 recognition of two stem loops in the sgRNA scaffold ( magenta ). These complexes were targeted to the IL8 or CXCL2 enhancers and promoters (highlights) via different sgRNA pools in HeLa. The position of individual sgRNAs used for CRISPRa is shown in more detail in Fig EV1 . Left bar graphs: Wild‐type HeLa cells were transiently transfected with different combinations of plasmids encoding the “dead” Cas9‐VP64 fusion protein, the MS2‐p65‐HSF1 fusion protein, and empty sgRNA vector or versions containing sgRNAs targeting the IL8 enhancer or promoter. Twenty‐four hours post‐transfection, cells were lysed and total RNA was analyzed for expression changes of the indicated genes compared to samples carrying dCas9‐VP64 and MS2‐p65‐HSF1 fusions, but no sgRNAs. Right bar graphs: The same experiments were performed using sgRNAs targeting the CXCL2 enhancer and promoter. Data information: All data are from four independent transfections. Shown are mean values ± SEM. P ‐values are derived from unpaired t ‐tests comparing every condition against cells expressing all transactivators but lacking sgRNAs (first lane in each graph). Only significant differences are marked by asterisks.

    Journal: The EMBO Journal

    Article Title: Distinct IL‐1α‐responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

    doi: 10.15252/embj.2019101533

    Figure Lengend Snippet: Heterologous activation of the IL8 enhancer triggers IL8 and CXCL1 expression A CRISPR activation (CRISPRa) strategy was applied to test enhancer functions individually. This approach involves a “dead” Cas9 ( blue ) and VP64 ( green ) fusion protein that recruits the NF‐κB ( orange ) and HSF1 ( red ) transactivation domains via MS2 recognition of two stem loops in the sgRNA scaffold ( magenta ). These complexes were targeted to the IL8 or CXCL2 enhancers and promoters (highlights) via different sgRNA pools in HeLa. The position of individual sgRNAs used for CRISPRa is shown in more detail in Fig EV1 . Left bar graphs: Wild‐type HeLa cells were transiently transfected with different combinations of plasmids encoding the “dead” Cas9‐VP64 fusion protein, the MS2‐p65‐HSF1 fusion protein, and empty sgRNA vector or versions containing sgRNAs targeting the IL8 enhancer or promoter. Twenty‐four hours post‐transfection, cells were lysed and total RNA was analyzed for expression changes of the indicated genes compared to samples carrying dCas9‐VP64 and MS2‐p65‐HSF1 fusions, but no sgRNAs. Right bar graphs: The same experiments were performed using sgRNAs targeting the CXCL2 enhancer and promoter. Data information: All data are from four independent transfections. Shown are mean values ± SEM. P ‐values are derived from unpaired t ‐tests comparing every condition against cells expressing all transactivators but lacking sgRNAs (first lane in each graph). Only significant differences are marked by asterisks.

    Article Snippet: For CHIP, the following antibodies were used: anti‐histone H3 (2 μg; Abcam; ab1791), anti‐NF‐κB p65 (3 μg; Santa Cruz; sc‐372), anti‐phospho‐Pol II (S5) (1.35 μg; Abcam; ab5131), H3K27ac (2 μg; Diagenode, pAb‐174‐050), H3K4me1 (2 μg; Abcam, ab8895), IgG (2 μg; Cell Signaling; 2729), and CTCF (4 μl; Millipore, 07‐729).

    Techniques: Activation Assay, Expressing, CRISPR, Transfection, Plasmid Preparation, Derivative Assay

    Intronic RNA FISH reveals reduced concomitant biallelic and colocalizing chemokine expression in enhancer‐mutant HeLa Representative triple RNA FISH images from HeLa cells ± IL‐1α stimulation for 60 min. Mature mRNAs ( IL8 , β‐actin ; red ) and intronic RNAs ( CXCL2 , purple ; IL8, green ) are detected against nuclei stained with Hoechst 33342 ( blue ). Typical foci marking individually labeled IL8 / CXCL2 transcription sites or merged signals indicating co‐transcription and spatial proximity are enlarged ( inset ). Scale bar: 10 μm. Quantification of RNA FISH signals from parental (wt), control (vector), p65‐deletion (Δp65 eIL8 and Δp65 eCXCL2 ), or p65‐knockout (Δ RELA ) HeLa lines ± IL‐1α stimulation for 60 min. Negative controls (neg ctrl) indicate samples from IL‐1α‐stimulated control cells in which RNA FISH was performed using pre‐amplifier, amplifier, and label probe mixes, but omitting the specific probe sets for IL8 or CXCL2 . These samples were used to define unspecific signals. Data from three independent experiments are pooled and plotted. The box plots show the distributions of FISH signals. Boundaries of the box indicate the 25 th /75 th percentiles, black lines mark medians, and colored lines mark means, respectively. Whiskers (error bars) indicate the 10 th /90 th percentiles, and circles mark all remaining outliers. The data from panel (B) were used to separately quantify the fraction of cells with mono‐ or biallelic IL8 or CXCL2 intronic RNA expression ( purple, green, blue colors ), as well as the extent of colocalizing (overlapping) intronic RNA FISH signals in individual cells, indicating simultaneously activated transcription sites on the same allele ( yellow colors ). The total numbers of cells analyzed are shown above each bar. Data are depicted relative to the total number of analyzed cells. * P

    Journal: The EMBO Journal

    Article Title: Distinct IL‐1α‐responsive enhancers promote acute and coordinated changes in chromatin topology in a hierarchical manner

    doi: 10.15252/embj.2019101533

    Figure Lengend Snippet: Intronic RNA FISH reveals reduced concomitant biallelic and colocalizing chemokine expression in enhancer‐mutant HeLa Representative triple RNA FISH images from HeLa cells ± IL‐1α stimulation for 60 min. Mature mRNAs ( IL8 , β‐actin ; red ) and intronic RNAs ( CXCL2 , purple ; IL8, green ) are detected against nuclei stained with Hoechst 33342 ( blue ). Typical foci marking individually labeled IL8 / CXCL2 transcription sites or merged signals indicating co‐transcription and spatial proximity are enlarged ( inset ). Scale bar: 10 μm. Quantification of RNA FISH signals from parental (wt), control (vector), p65‐deletion (Δp65 eIL8 and Δp65 eCXCL2 ), or p65‐knockout (Δ RELA ) HeLa lines ± IL‐1α stimulation for 60 min. Negative controls (neg ctrl) indicate samples from IL‐1α‐stimulated control cells in which RNA FISH was performed using pre‐amplifier, amplifier, and label probe mixes, but omitting the specific probe sets for IL8 or CXCL2 . These samples were used to define unspecific signals. Data from three independent experiments are pooled and plotted. The box plots show the distributions of FISH signals. Boundaries of the box indicate the 25 th /75 th percentiles, black lines mark medians, and colored lines mark means, respectively. Whiskers (error bars) indicate the 10 th /90 th percentiles, and circles mark all remaining outliers. The data from panel (B) were used to separately quantify the fraction of cells with mono‐ or biallelic IL8 or CXCL2 intronic RNA expression ( purple, green, blue colors ), as well as the extent of colocalizing (overlapping) intronic RNA FISH signals in individual cells, indicating simultaneously activated transcription sites on the same allele ( yellow colors ). The total numbers of cells analyzed are shown above each bar. Data are depicted relative to the total number of analyzed cells. * P

    Article Snippet: For CHIP, the following antibodies were used: anti‐histone H3 (2 μg; Abcam; ab1791), anti‐NF‐κB p65 (3 μg; Santa Cruz; sc‐372), anti‐phospho‐Pol II (S5) (1.35 μg; Abcam; ab5131), H3K27ac (2 μg; Diagenode, pAb‐174‐050), H3K4me1 (2 μg; Abcam, ab8895), IgG (2 μg; Cell Signaling; 2729), and CTCF (4 μl; Millipore, 07‐729).

    Techniques: Fluorescence In Situ Hybridization, Expressing, Mutagenesis, Staining, Labeling, Plasmid Preparation, Knock-Out, RNA Expression

    Effect of the reactive oxygen species (ROS) inhibitor N-acetylcysteine (NAC) on the nuclear factor (NF)-κB signaling pathway in xanthohumol (Xn)-treated AGS cells. Cells were pre-treated with the ROS inhibitor NAC (5 mM) for 1 h, and then treated with Xn (20 µ M) for 24 h; they were then harvested and lysed to measure NF-κB signaling proteins through western blotting. (A-C) Expression of IκBα and p-IκBα protein; (D-F) Expression of nuclear and cytosolic p65 protein. Histone H3 served as the nuclear loading control, GAPDH served as the cytosolic loading control. Data are expressed as mean ± standard error of the mean. n=3. **P

    Journal: Oncology Reports

    Article Title: Xanthohumol, a prenylated flavonoid from Hops, exerts anticancer effects against gastric cancer in vitro

    doi: 10.3892/or.2018.6723

    Figure Lengend Snippet: Effect of the reactive oxygen species (ROS) inhibitor N-acetylcysteine (NAC) on the nuclear factor (NF)-κB signaling pathway in xanthohumol (Xn)-treated AGS cells. Cells were pre-treated with the ROS inhibitor NAC (5 mM) for 1 h, and then treated with Xn (20 µ M) for 24 h; they were then harvested and lysed to measure NF-κB signaling proteins through western blotting. (A-C) Expression of IκBα and p-IκBα protein; (D-F) Expression of nuclear and cytosolic p65 protein. Histone H3 served as the nuclear loading control, GAPDH served as the cytosolic loading control. Data are expressed as mean ± standard error of the mean. n=3. **P

    Article Snippet: Antibodies against Bcl-2 (rabbit polyclonal antibody, dilution 1:1,000; cat. no. ab194583), Bax (rabbit monoclonal antibody, dilution 1:1,000; cat. no. ab32503), p-IκBα (rabbit monoclonal antibody, dilution 1:1,000; cat. no. ab133462), IκBα (rabbit monoclonal antibody, dilution 1:1,000; cat. no. ab32518), p65 (rabbit polyclonal antibody, dilution 1:1,000; cat. no. ab16502), histone H3 (rabbit polyclonal antibody, dilution 1:1,000; cat. no. ab1791) and GAPDH (rabbit polyclonal antibody, dilution 1:1,000; cat. no. ab9485) were obtained from Abcam (Cambridge, UK).

    Techniques: Western Blot, Expressing

    Effect of xanthohumol (Xn) on the nuclear factor (NF)-κB signaling pathway in AGS cells. Cells were treated with different concentrations of Xn (0–20 µ M) for 24 h, then harvested and lysed to measure NF-κB signaling proteins through western blotting. (A-C) Effects of Xn on IκBα and p-IκBα expression. (D-F) Effect of Xn on nuclear and cytosolic p65 expression. Histone H3 served as the nuclear loading control and GAPDH served as the cytosolic loading control. Data are expressed as mean ± standard error of the mean. n=3. *P

    Journal: Oncology Reports

    Article Title: Xanthohumol, a prenylated flavonoid from Hops, exerts anticancer effects against gastric cancer in vitro

    doi: 10.3892/or.2018.6723

    Figure Lengend Snippet: Effect of xanthohumol (Xn) on the nuclear factor (NF)-κB signaling pathway in AGS cells. Cells were treated with different concentrations of Xn (0–20 µ M) for 24 h, then harvested and lysed to measure NF-κB signaling proteins through western blotting. (A-C) Effects of Xn on IκBα and p-IκBα expression. (D-F) Effect of Xn on nuclear and cytosolic p65 expression. Histone H3 served as the nuclear loading control and GAPDH served as the cytosolic loading control. Data are expressed as mean ± standard error of the mean. n=3. *P

    Article Snippet: Antibodies against Bcl-2 (rabbit polyclonal antibody, dilution 1:1,000; cat. no. ab194583), Bax (rabbit monoclonal antibody, dilution 1:1,000; cat. no. ab32503), p-IκBα (rabbit monoclonal antibody, dilution 1:1,000; cat. no. ab133462), IκBα (rabbit monoclonal antibody, dilution 1:1,000; cat. no. ab32518), p65 (rabbit polyclonal antibody, dilution 1:1,000; cat. no. ab16502), histone H3 (rabbit polyclonal antibody, dilution 1:1,000; cat. no. ab1791) and GAPDH (rabbit polyclonal antibody, dilution 1:1,000; cat. no. ab9485) were obtained from Abcam (Cambridge, UK).

    Techniques: Western Blot, Expressing

    Expression and distribution of HMGB1, NF-κB p65 and OX42 in the epileptic brain tissues of patients with refractory epilepsy. HMGB1, high-mobility group box-1; p-, phosphorylated; NF-κB, nuclear factor-κB; EP, refractory epilepsy group. Red and purple arrows indicate the protein distribution of HMGB1 and NF-κB p65, respectively. Magnification, ×200.

    Journal: Molecular Medicine Reports

    Article Title: HMGB1 mediates microglia activation via the TLR4/NF-κB pathway in coriaria lactone induced epilepsy

    doi: 10.3892/mmr.2018.8485

    Figure Lengend Snippet: Expression and distribution of HMGB1, NF-κB p65 and OX42 in the epileptic brain tissues of patients with refractory epilepsy. HMGB1, high-mobility group box-1; p-, phosphorylated; NF-κB, nuclear factor-κB; EP, refractory epilepsy group. Red and purple arrows indicate the protein distribution of HMGB1 and NF-κB p65, respectively. Magnification, ×200.

    Article Snippet: Sections were then incubated with primary antibodies: Anti-HMGB1 (cat. no. ab18256, 1:100; Abcam, Cambridge, UK), anti-NF-κB p65 (cat. no. ab16502, 1:5,000; Abcam) and anti-OX42 (cat. no. ab1211, 1:500; Abcam) in 3% bovine albumin serum in PBS at 4°C overnight.

    Techniques: Expressing

    Expression level of HMGB1, TLR4, RAGE, NF-κB p65 and iNOS in the brain tissues of the control and the EP group, as detected by western blotting. HMGB1, high-mobility group box-1; NF-κB, nuclear factor-κB; EP, refractory epilepsy group; TLR4, toll-like receptor 4; RAGE, receptor for advanced glycation end products; iNOS, inducible nitric oxide synthase.

    Journal: Molecular Medicine Reports

    Article Title: HMGB1 mediates microglia activation via the TLR4/NF-κB pathway in coriaria lactone induced epilepsy

    doi: 10.3892/mmr.2018.8485

    Figure Lengend Snippet: Expression level of HMGB1, TLR4, RAGE, NF-κB p65 and iNOS in the brain tissues of the control and the EP group, as detected by western blotting. HMGB1, high-mobility group box-1; NF-κB, nuclear factor-κB; EP, refractory epilepsy group; TLR4, toll-like receptor 4; RAGE, receptor for advanced glycation end products; iNOS, inducible nitric oxide synthase.

    Article Snippet: Sections were then incubated with primary antibodies: Anti-HMGB1 (cat. no. ab18256, 1:100; Abcam, Cambridge, UK), anti-NF-κB p65 (cat. no. ab16502, 1:5,000; Abcam) and anti-OX42 (cat. no. ab1211, 1:500; Abcam) in 3% bovine albumin serum in PBS at 4°C overnight.

    Techniques: Expressing, Western Blot

    Western blotting of protein expression levels in HM cells. (A) CL significantly increases the levels of HMGB1, TLR4, RAGE, NF-κB p65 and iNOS in HM cells. (B) Overexpression of HMGB1 this increased further, and inhibition of HMGB1 by siRNA blocked the function of CL to HM cells. HMGB1, high-mobility group box-1; NF-κB, nuclear factor-κB; EP, refractory epilepsy group; TLR4, toll-like receptor 4; RAGE, receptor for advanced glycation end products; iNOS, inducible nitric oxide synthase; p-phosphorylated; siRNA, small interfering RNA; NC, negative control; CL, cariaria lactone.

    Journal: Molecular Medicine Reports

    Article Title: HMGB1 mediates microglia activation via the TLR4/NF-κB pathway in coriaria lactone induced epilepsy

    doi: 10.3892/mmr.2018.8485

    Figure Lengend Snippet: Western blotting of protein expression levels in HM cells. (A) CL significantly increases the levels of HMGB1, TLR4, RAGE, NF-κB p65 and iNOS in HM cells. (B) Overexpression of HMGB1 this increased further, and inhibition of HMGB1 by siRNA blocked the function of CL to HM cells. HMGB1, high-mobility group box-1; NF-κB, nuclear factor-κB; EP, refractory epilepsy group; TLR4, toll-like receptor 4; RAGE, receptor for advanced glycation end products; iNOS, inducible nitric oxide synthase; p-phosphorylated; siRNA, small interfering RNA; NC, negative control; CL, cariaria lactone.

    Article Snippet: Sections were then incubated with primary antibodies: Anti-HMGB1 (cat. no. ab18256, 1:100; Abcam, Cambridge, UK), anti-NF-κB p65 (cat. no. ab16502, 1:5,000; Abcam) and anti-OX42 (cat. no. ab1211, 1:500; Abcam) in 3% bovine albumin serum in PBS at 4°C overnight.

    Techniques: Western Blot, Expressing, Over Expression, Inhibition, Small Interfering RNA, Negative Control

    12-HETE promotes the activation and nuclear translocation of NF-κB through the ILK pathway. Notes: ( A ) 1 µM 12-HETE treatment led to increased levels of phosphorylated NF-κB p65 but was antagonized by the knockdown of ILK. ( B ) 1 µM 12-HETE significantly increased the protein level of nuclear NF-κB p65 and decreased the protein level of NF-κB p65 in the cytoplasm via ILK. ( C ) The 12-HETE-induced phosphorylation of IKBa was mediated by ILK in ovarian cancer cells. All values are represented as the mean±SEM from three or more independent batches of cells (* P

    Journal: Cancer Management and Research

    Article Title: 12-HETE facilitates cell survival by activating the integrin-linked kinase/NF-κB pathway in ovarian cancer

    doi: 10.2147/CMAR.S180334

    Figure Lengend Snippet: 12-HETE promotes the activation and nuclear translocation of NF-κB through the ILK pathway. Notes: ( A ) 1 µM 12-HETE treatment led to increased levels of phosphorylated NF-κB p65 but was antagonized by the knockdown of ILK. ( B ) 1 µM 12-HETE significantly increased the protein level of nuclear NF-κB p65 and decreased the protein level of NF-κB p65 in the cytoplasm via ILK. ( C ) The 12-HETE-induced phosphorylation of IKBa was mediated by ILK in ovarian cancer cells. All values are represented as the mean±SEM from three or more independent batches of cells (* P

    Article Snippet: The antibodies against Histone 1.2 (ab17677) and NF-κB p65 (phospho-S536; ab86299) were purchased from Abcam (Shanghai, China).

    Techniques: Activation Assay, Translocation Assay

    Inhibitory effects of hypericin-photodynamic therapy on NF-κB activation in MH7A cells. Cells were treated with HYP (0–4 μM) and PDT. NF-κB pathway proteins and mRNA were measured with Western blot and qRT-PCR, with β-actin used as an internal control. (A) Western blot data is shown for p-NF-κB p65, NF-κB p65 and p-IκBα and densitometric analysis performed using ImageJ software. Relative quantities of p-NF-κB p65 (B), NF-κB p65 (C) and p-IκBα (D) were normalized to untreated cells (0 μM HYP; 100%). (E) NF-κB p65 mRNA was assessed using qRT-PCR. Gene expression was normalized to GAPDH. Data are means±SD (n=3); *** P

    Journal: Iranian Journal of Basic Medical Sciences

    Article Title: Hypericin-photodynamic therapy inhibits proliferation and induces apoptosis in human rheumatoid arthritis fibroblast-like synoviocytes cell line MH7A

    doi: 10.22038/IJBMS.2018.23871.5991

    Figure Lengend Snippet: Inhibitory effects of hypericin-photodynamic therapy on NF-κB activation in MH7A cells. Cells were treated with HYP (0–4 μM) and PDT. NF-κB pathway proteins and mRNA were measured with Western blot and qRT-PCR, with β-actin used as an internal control. (A) Western blot data is shown for p-NF-κB p65, NF-κB p65 and p-IκBα and densitometric analysis performed using ImageJ software. Relative quantities of p-NF-κB p65 (B), NF-κB p65 (C) and p-IκBα (D) were normalized to untreated cells (0 μM HYP; 100%). (E) NF-κB p65 mRNA was assessed using qRT-PCR. Gene expression was normalized to GAPDH. Data are means±SD (n=3); *** P

    Article Snippet: Antibodies against caspase-8, caspase-9, cleaved caspase-9, poly-ADP-ribose polymerase (PARP), cleaved PARP, p-NF-κB p65, NF-κB p65, p-IκBα, and β-actin were purchased from Cell Signaling Technology (Boston, MA, USA).

    Techniques: Activation Assay, Western Blot, Quantitative RT-PCR, Software, Expressing

    Treatment with AG1478 reduced levels of lipopolysaccharide (LPS)-induced hepatic stellate cell (HSC) inflammatory cytokines. Hepatic stellate cells (HSCs) were pretreated with AG1478 (2.5 μM and 5 μM) for 2 h, and then exposed to lipopolysaccharide (LPS) (100 ng/mL) for the indicated times. ( A, B ) HSCs incubated with LPS for 24 h. Levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6, in the cell lysates were detected by Western blot ( A ). The figures in the columns show the normalized optical density (OD) for the data from three independent experiments ( B ). ( C, D ) HSCs incubated with LPS for 6 h. The mRNA levels of TNF-α ( C ) and IL-6 ( D ) were detected by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and normalized against β-actin. ( E–G ) Western blot analysis of IκBα ( E ), cytoplasm NFκB P65 (C-NFκB) ( F ) and nuclear NFκB P65(N-NFκB) ( G ) levels in HSCs incubated with LPS for 1h. GAPDH was used as a loading control for IκBα/C-NFκB and laminin B as loading control for N-NFκB. ns – not significant vs. Ctrl group; ** P

    Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

    Article Title: Inhibition of Epidermal Growth Factor Receptor (EGFR) Reduces Lipopolysaccharide (LPS)-Induced Activation and Inflammatory Cytokines in Hepatic Stellate Cells In Vitro

    doi: 10.12659/MSM.909901

    Figure Lengend Snippet: Treatment with AG1478 reduced levels of lipopolysaccharide (LPS)-induced hepatic stellate cell (HSC) inflammatory cytokines. Hepatic stellate cells (HSCs) were pretreated with AG1478 (2.5 μM and 5 μM) for 2 h, and then exposed to lipopolysaccharide (LPS) (100 ng/mL) for the indicated times. ( A, B ) HSCs incubated with LPS for 24 h. Levels of tumor necrosis factor (TNF)-α and interleukin (IL)-6, in the cell lysates were detected by Western blot ( A ). The figures in the columns show the normalized optical density (OD) for the data from three independent experiments ( B ). ( C, D ) HSCs incubated with LPS for 6 h. The mRNA levels of TNF-α ( C ) and IL-6 ( D ) were detected by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and normalized against β-actin. ( E–G ) Western blot analysis of IκBα ( E ), cytoplasm NFκB P65 (C-NFκB) ( F ) and nuclear NFκB P65(N-NFκB) ( G ) levels in HSCs incubated with LPS for 1h. GAPDH was used as a loading control for IκBα/C-NFκB and laminin B as loading control for N-NFκB. ns – not significant vs. Ctrl group; ** P

    Article Snippet: Primary antibodies to epidermal growth factor receptor (EGFR), phospho-EGFR (p-EGFR), Tyr835, TLR4, tumor necrosis factor (TNF)-α, interleukin (IL)-6, IκB-α (an inhibitor of NF-κB) and NFκB P65 were obtained from Cell Signaling Technology (Danvers, MA, USA).

    Techniques: Incubation, Western Blot, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR

    Synergistic inhibitory effects of AC and the NF-κB signaling pathway inhibitor JSH-23 on colorectal cancer cells. (A) CCK-8 assay was performed to detect the viability of HCT116 cells treated with AC and JSH-23 for 24, 48 and 72 h. (B) Cell cycle of HCT116 cells treated with AC and JSH-23 for 24 h was assessed using flow cytometry. (C) Statistical analysis of cell cycle of HCT116 cells co-treated with AC and JSH-23 for 24 h. (D) Cell cycle of HCT116 cells co-treated with AC and JSH-23 for 72 h was measured by flow cytometry. (E) Quantification of cell cycle of HCT116 cells co-treated with AC and JSH-23 for 72 h. (F) Cell apoptosis of HCT116 cells co-treated with AC and JSH-23 for 24 h was determined using flow cytometry. (G) Cell apoptosis of HCT116 cells co-treated with AC and JSH-23 for 72 h was detected by flow cytometry. (H) Statistical analysis of cell apoptosis of HCT116 cells co-treated with AC and JSH-23 for 24 h. (I) Statistical analysis of cell apoptosis of HCT116 cells co-treated with AC and JSH-23 for 72 h. (J) The phosphorylation of IκBα and P65 was measured by western blotting. Data are expressed as mean ± standard deviation from three independent experiments. * P

    Journal: International Journal of Molecular Medicine

    Article Title: Asiaticoside suppresses cell proliferation by inhibiting the NF-κB signaling pathway in colorectal cancer

    doi: 10.3892/ijmm.2020.4688

    Figure Lengend Snippet: Synergistic inhibitory effects of AC and the NF-κB signaling pathway inhibitor JSH-23 on colorectal cancer cells. (A) CCK-8 assay was performed to detect the viability of HCT116 cells treated with AC and JSH-23 for 24, 48 and 72 h. (B) Cell cycle of HCT116 cells treated with AC and JSH-23 for 24 h was assessed using flow cytometry. (C) Statistical analysis of cell cycle of HCT116 cells co-treated with AC and JSH-23 for 24 h. (D) Cell cycle of HCT116 cells co-treated with AC and JSH-23 for 72 h was measured by flow cytometry. (E) Quantification of cell cycle of HCT116 cells co-treated with AC and JSH-23 for 72 h. (F) Cell apoptosis of HCT116 cells co-treated with AC and JSH-23 for 24 h was determined using flow cytometry. (G) Cell apoptosis of HCT116 cells co-treated with AC and JSH-23 for 72 h was detected by flow cytometry. (H) Statistical analysis of cell apoptosis of HCT116 cells co-treated with AC and JSH-23 for 24 h. (I) Statistical analysis of cell apoptosis of HCT116 cells co-treated with AC and JSH-23 for 72 h. (J) The phosphorylation of IκBα and P65 was measured by western blotting. Data are expressed as mean ± standard deviation from three independent experiments. * P

    Article Snippet: The following primary antibodies were used for incubation: Anti-NF-kB P65 (1:1,000; cat. no. ab16502; Abcam), anti-phosphorylated (p)-P65 (1:1,500; cat. no. ab86299; Abcam), anti-caspase-3 (1:1,000; cat. no. ab4051, Abcam), anti-cleaved caspase-3 (1:1,000; cat. no. ab32042; Abcam), anti-caspase-9 (1:1,000; cat. no. ab32539; Abcam), anti-cleaved caspase-9 (1:1,000; cat. no. ab32539; Abcam), anti-Bcl-2 (1:1,000; cat. no. ab32124; Abcam), anti-Bax (1:1,000; cat. no. ab32503; Abcam), anti-CDK4 (1:1,000; cat. no. ab199728, Abcam), anti-Cyclin D1 (1:2,000; cat. no. ab205718; Abcam), anti-IκBα (1:1,000; cat. no. 9242; Cell Signaling Technology, Inc.), anti-p-IκBα (1:1,000; cat. no. 2859; Cell Signaling Technology, Inc.), anti-Histone H3 (1:3,000; cat. no. ab1791; Abcam) and anti-β-actin (1:5,000; cat. no. ab8227; Abcam).

    Techniques: CCK-8 Assay, Flow Cytometry, Western Blot, Standard Deviation

    Effect of AC on the NF-κB signaling pathway. (A) mRNA and (B) protein expression levels of the NF-κB signaling pathway-related molecules IκBα, p-IκBα, P65 and p-P65 in HCT116 cells treated with AC for 72 h. (C) Quantification of the western blots. (D) Nuclear translocation of P65 detected by western blotting in HCT116 cells treated with 2 µ M AC for 72 h. (E) Quantification of the western blots. Data are expressed as mean ± standard deviation from three independent experiments. * P

    Journal: International Journal of Molecular Medicine

    Article Title: Asiaticoside suppresses cell proliferation by inhibiting the NF-κB signaling pathway in colorectal cancer

    doi: 10.3892/ijmm.2020.4688

    Figure Lengend Snippet: Effect of AC on the NF-κB signaling pathway. (A) mRNA and (B) protein expression levels of the NF-κB signaling pathway-related molecules IκBα, p-IκBα, P65 and p-P65 in HCT116 cells treated with AC for 72 h. (C) Quantification of the western blots. (D) Nuclear translocation of P65 detected by western blotting in HCT116 cells treated with 2 µ M AC for 72 h. (E) Quantification of the western blots. Data are expressed as mean ± standard deviation from three independent experiments. * P

    Article Snippet: The following primary antibodies were used for incubation: Anti-NF-kB P65 (1:1,000; cat. no. ab16502; Abcam), anti-phosphorylated (p)-P65 (1:1,500; cat. no. ab86299; Abcam), anti-caspase-3 (1:1,000; cat. no. ab4051, Abcam), anti-cleaved caspase-3 (1:1,000; cat. no. ab32042; Abcam), anti-caspase-9 (1:1,000; cat. no. ab32539; Abcam), anti-cleaved caspase-9 (1:1,000; cat. no. ab32539; Abcam), anti-Bcl-2 (1:1,000; cat. no. ab32124; Abcam), anti-Bax (1:1,000; cat. no. ab32503; Abcam), anti-CDK4 (1:1,000; cat. no. ab199728, Abcam), anti-Cyclin D1 (1:2,000; cat. no. ab205718; Abcam), anti-IκBα (1:1,000; cat. no. 9242; Cell Signaling Technology, Inc.), anti-p-IκBα (1:1,000; cat. no. 2859; Cell Signaling Technology, Inc.), anti-Histone H3 (1:3,000; cat. no. ab1791; Abcam) and anti-β-actin (1:5,000; cat. no. ab8227; Abcam).

    Techniques: Expressing, Western Blot, Translocation Assay, Standard Deviation

    Proteasome inhibitors act directly on the HIV LTR to activate HIV in an NF-κB-dependent manner. (A) Chronically HIV-infected J-Lat 10.6 cells were treated with DMSO, bortezomib, or ixazomib and assessed for GFP expression over time. Depicted are means (±SD) of the results of four independent experiments. (B) J-Lat 10.6 cells treated as for panel A were assessed for HIV p55 Gag expression by Western blotting. (C) Jurkat CD4 + T cells transfected with an HIV-1 LTR firefly luciferase reporter construct were treated with control, bortezomib, or ixazomib, and luciferase activity was measured and normalized to that of Renilla luciferase. Depicted is fold luciferase induction relative to that of control-treated cells (means and SD of the results of five independent experiments). (D) Jurkat CD4 + T cells transfected with the HIV LTR luciferase construct with or without deletions in the NF-κB-binding site (ΔKb) were treated with control, bortezomib, or ixazomib and assessed for luciferase activity, normalized to that of Renilla luciferase. Depicted is fold luciferase induction relative to that of control-treated cells (means and SD of the results of two independent experiments). (E) J-Lat 10.6 cells were treated with control, bortezomib, or ixazomib for 6 or 24 h, as indicated, and expression of NF-κB p65, Ser536 phospho-NF-κB, IκBα, and Ser32 phospho-IκBα was assessed by Western blot analysis. *, P

    Journal: Journal of Virology

    Article Title: HIV Protease-Generated Casp8p41, When Bound and Inactivated by Bcl2, Is Degraded by the Proteasome

    doi: 10.1128/JVI.00037-18

    Figure Lengend Snippet: Proteasome inhibitors act directly on the HIV LTR to activate HIV in an NF-κB-dependent manner. (A) Chronically HIV-infected J-Lat 10.6 cells were treated with DMSO, bortezomib, or ixazomib and assessed for GFP expression over time. Depicted are means (±SD) of the results of four independent experiments. (B) J-Lat 10.6 cells treated as for panel A were assessed for HIV p55 Gag expression by Western blotting. (C) Jurkat CD4 + T cells transfected with an HIV-1 LTR firefly luciferase reporter construct were treated with control, bortezomib, or ixazomib, and luciferase activity was measured and normalized to that of Renilla luciferase. Depicted is fold luciferase induction relative to that of control-treated cells (means and SD of the results of five independent experiments). (D) Jurkat CD4 + T cells transfected with the HIV LTR luciferase construct with or without deletions in the NF-κB-binding site (ΔKb) were treated with control, bortezomib, or ixazomib and assessed for luciferase activity, normalized to that of Renilla luciferase. Depicted is fold luciferase induction relative to that of control-treated cells (means and SD of the results of two independent experiments). (E) J-Lat 10.6 cells were treated with control, bortezomib, or ixazomib for 6 or 24 h, as indicated, and expression of NF-κB p65, Ser536 phospho-NF-κB, IκBα, and Ser32 phospho-IκBα was assessed by Western blot analysis. *, P

    Article Snippet: The following antibodies were used for immunoblotting or immunoprecipitation: anti-HA peroxidase (3F10) rat monoclonal antibody (catalog number 12013819001; Roche, Indianapolis, IN), mouse anti-caspase 8 antibody (received from Marcus E. Peter, Northwestern University, Chicago, IL), mouse monoclonal anti-ubiquitin (P4D1) antibody (sc-8017; Santa Cruz), anti-Bcl-2 (C21) rabbit polyclonal IgG (catalog number sc-783; Santa Cruz), monoclonal anti-HIV p24 (catalog number 530; NIH AIDS Reagent Program), anti-GAPDH (glyceraldehyde-3-phosphate dehydrogenase) (14C10) rabbit MAb (catalog number 2118; Cell Signaling), histone deacetylase (HDAC) antibody sampler kit (catalog number 9928; Cell Signaling), anti-phospho-NF-κB-p65 (Ser 536) rabbit MAb (catalog number 3033; Cell Signaling), anti-NF-κBp65 (D14E12) rabbit MAb (catalog number 8242; Cell Signaling), anti-phospho-IκBα (Ser32) (14D4) rabbit MAb (catalog number 2859; Cell Signaling), anti-IκBα (L35A5) mouse MAb (amino-terminal antigen; catalog number 4814; Cell Signaling), anti-cleaved caspase 3 (Asp175) antibody (catalog number 9661; Cell Signaling), and mouse anti-human PARP (catalog number 556494; BD Pharmingen).

    Techniques: Activated Clotting Time Assay, Infection, Expressing, Western Blot, Transfection, Luciferase, Construct, Activity Assay, Binding Assay

    NF-κB/p65 is responsible for the downregulation of MMP-9 and Bfl-1 mediated by fordin in U-2 OS and HepG2 cells. The expression of p65 (phosphorylated and total), MMP-9 and Bfl-1 were determined by western blot analysis in the various treated cells and normalized to β-actin or Histone H3. (A) U-2 OS and (B) HepG2 cells were incubated with various concentrations of fordin for 24 h; * P

    Journal: International Journal of Oncology

    Article Title: Fordin: A novel type I ribosome inactivating protein from Vernicia fordii modulates multiple signaling cascades leading to anti-invasive and pro-apoptotic effects in cancer cells in vitro

    doi: 10.3892/ijo.2018.4470

    Figure Lengend Snippet: NF-κB/p65 is responsible for the downregulation of MMP-9 and Bfl-1 mediated by fordin in U-2 OS and HepG2 cells. The expression of p65 (phosphorylated and total), MMP-9 and Bfl-1 were determined by western blot analysis in the various treated cells and normalized to β-actin or Histone H3. (A) U-2 OS and (B) HepG2 cells were incubated with various concentrations of fordin for 24 h; * P

    Article Snippet: The other primary antibodies were anti-Bcl-2-related protein A1 (Bfl-1; 1:200, cat. no. ab45413), anti-MMP-2 (1:500, cat. no. ab92536), anti-MMP-9 (1:500, cat. no. ab76003), anti-p65 [(phosphorylated S536; 1:1,000, cat. no. ab76302) and total (1:1,000, cat. no. ab76311)], anti-IKKα (1:1,000, cat. no. ab32518), anti-IKKβ (1:1,000, cat. no. ab124957), and anti-IκB [(phosphorylated S36; 1:1,000, cat. no. ab133462) and total (1:1,000, cat. no. ab32518)], all of which were purchased from Abcam (Cambridge, MA, USA).

    Techniques: Expressing, Western Blot, Incubation