Journal: Molecular Medicine

Article Title: Targeting Activation of Specific NF-?B Subunits Prevents Stress-Dependent Atherothrombotic Gene Expression

doi: 10.2119/molmed.2012.00282

Figure Lengend Snippet: Norepinephrine and exposure to stress induced NF-κB activation in vitro and ex vivo. NE induced NF-κB activation and subsequent gene expression is mediated by differential composition of NF-κB heterodimers. (A) THP-1-cells were incubated with NE for the indicated times and RT-PCR was performed to determine three types of transcription: ICAM-1, TF and MnSOD. (B) NF-κB binding activity at binding sites of ICAM-1, TF and MnSOD genes measured by EMSA with nuclear extracts from THP-1 cells incubated with NE for 15 min (Control = unstimulated; NE = norepinephrine). (C) Chromatin immunoprecipitation with antibodies to p50, p65, cRel was performed as described in Methods using unstimulated and NE-induced THP-1 cellsan was followed by PCR amplification of NF-κB binding sites in ICAM-1, TF and MnSOD genes. (D) THP-1 cells were left untreated or incubated with antisense or scrambled ODNs for NF-κB p50, p65 or cRel as described in Materials and Methods. Four mRNA levels—ICAM-1, TF, MnSOD and β-actin—were analyzed using RT-PCR. All experiments were repeated at least three times with identical results. (E) pBMC were either isolated from pooled total blood from two C57Bl/6 mice/measure and cultured for 24 h before either left untreated or stimulated with 10 nmol/L NE for 15 min or (1,2) from total blood of five ApoE−/− mice/group, whereas part of the mice were untreated (3) or underwent a 20 min immobilization stress in the absence (4) or presence of adrenergic inhibitors (5). Chromatin immunoprecipitation was performed followed by PCR amplification of NF-κB binding site in the murine ICAM-1 promoter. Total chromatin served as positive control. The experiment was repeated twice with identical results. (F) pBMC were isolated from pooled total blood derived from five ApoE−/− mice/group and analyzed for ICAM-1, TF, MnSOD and β-actin mRNA levels using RT-PCR. The experiment was repeated twice with identical results.

Article Snippet: Membranes were incubated with anti-NF-κB p50 (Epitomics, Burlingame, CA, USA), anti-NF-κB p65 and anti-NF-κB cRel (Rockland Im-munochemicals, Gilbertsville, PA, USA) antibodies (1:500 dilution).

Techniques: Activation Assay, In Vitro, Ex Vivo, Expressing, Incubation, Reverse Transcription Polymerase Chain Reaction, Binding Assay, Activity Assay, Chromatin Immunoprecipitation, Amplification, Isolation, Cell Culture, Positive Control, Derivative Assay

Journal: Molecular Medicine

Article Title: Targeting Activation of Specific NF-?B Subunits Prevents Stress-Dependent Atherothrombotic Gene Expression

doi: 10.2119/molmed.2012.00282

Figure Lengend Snippet: Effects of PKC, p38MAPK or PI3K pathway inhibition on NF-κB subunits nuclear translocation in vitro: THP-1 cells were preincubated for 45 min in the absence or presence of the PKC, p38MAPK or PI3K inhibitor before treatment with NE for 15 min. (A) Nuclear extracts of THP-1 cells were immunoblotted with antibodies to p50, p65, cRel and histone-1. The signal intensity was evaluated and is summarized in the bargraphs. Statistical significant differences are indicated (*p < 0.05). Data represent the mean ± SD (n = 5). (B) The recruitment of p50, p65 and cRel-subunits to the ICAM-1 promoter was determined using ChIP-assays. (C) NF-κB subunit activation in vivo. ApoE−/− mice were either untreated or underwent immobilization as described in Materials and Methods, in the absence or presence of the inhibitors of PKC, p38 MAPK or PI3K. Aortic sections were stained with antibodies to p50, p65and cRel (magnification: 20×). The experiment was repeated three times with identical results. (D) Target gene expression in aortic tissue: Frozen (top, bottom) or paraffin-embedded (middle) aortic sections were stained for ICAM-1, TF and MnSOD using magnification 20×. The experiment was repeated three times with identical results.

Article Snippet: Membranes were incubated with anti-NF-κB p50 (Epitomics, Burlingame, CA, USA), anti-NF-κB p65 and anti-NF-κB cRel (Rockland Im-munochemicals, Gilbertsville, PA, USA) antibodies (1:500 dilution).

Techniques: Inhibition, Translocation Assay, In Vitro, Activation Assay, In Vivo, Staining, Expressing

Journal: Molecular Medicine

Article Title: Targeting Activation of Specific NF-?B Subunits Prevents Stress-Dependent Atherothrombotic Gene Expression

doi: 10.2119/molmed.2012.00282

Figure Lengend Snippet: Selective inhibition of PI3K pathway and cRel leads to the decrease of atherosclerotic lesions in aortas of ApoE−/− mice exposed to the repeated transient stress. (A) ApoE−/− mice were exposed to repeated stress episodes as described in methods. Thereafter, total aortae from at least four ApoE−/− mice, untreated; stressed; pretreated with PKC, p38MAPK or PI3K inhibitor; were stained with oil red O and evaluated by one investigator blinded to the identities of the experimental groups. Statistically significant differences are indicated (**p < 0.01). (B) Untreated (same staining as in Figure 5A) or stressed ApoE−/− mice (same staining as in Figure 5A) were pretreated with siRNA for p50, p65, cRel. Aortae were stained with oil red O and evaluated by one investigator blinded to the identities of the experimental groups. Statistically significant differences are indicated (**p < 0.01).

Article Snippet: Membranes were incubated with anti-NF-κB p50 (Epitomics, Burlingame, CA, USA), anti-NF-κB p65 and anti-NF-κB cRel (Rockland Im-munochemicals, Gilbertsville, PA, USA) antibodies (1:500 dilution).

Techniques: Inhibition, Staining

Journal: Molecular Medicine

Article Title: Targeting Activation of Specific NF-?B Subunits Prevents Stress-Dependent Atherothrombotic Gene Expression

doi: 10.2119/molmed.2012.00282

Figure Lengend Snippet: cRel deficiency reduces atherosclerotic lesions and extracellular matrix changes in response to repeated transient stress. Apo E−/− mice underwent BM transplantation, receiving BM from either male Apo E−/−, C57Bl/6-wild-type or cRel−/− mice-donor mice. After 6 wks of reconstitution, mice either left untreated or subjected to repeated episodes of immobilization stress for 4 wks as described above. (A) Aortae of untreated and stressed ApoE−/− mice that had received BM from either ApoE−/− mice (lane 1, 2), C57BL6 mice (lane 3, 4) or cRel−/− mice (lane 5, 6) were stained with oil red O. The positive area was quantified using ImageJ software and is summarized in the bar graphs. Aortas from at least 3 mice per group were stained and quantified. Statistically significant differences are indicated by (*p < 0.05). Data represent the mean ± SD (n ≥ 3). The RT PCR at the bottom demonstrates the efficiency of the BM engraftment; RNA was isolated from the pBMC of the animals that underwent BM transplantation and assayed for NF-κB cRel transcripts. (B) Frozen aortic sections were stained with Movat pentachrome stain as described in methods. Magnification: 4×, 20×. Sections were analyzed with Image Pro software as described. The results are summarized in the bargraphs and statistical significant differences are indicated by (*p < 0.05; **p < 0.01). Data represent the mean ± SD (n = 3).

Article Snippet: Membranes were incubated with anti-NF-κB p50 (Epitomics, Burlingame, CA, USA), anti-NF-κB p65 and anti-NF-κB cRel (Rockland Im-munochemicals, Gilbertsville, PA, USA) antibodies (1:500 dilution).

Techniques: Transplantation Assay, Staining, Software, Reverse Transcription Polymerase Chain Reaction, Isolation

Journal: Molecular Medicine

Article Title: Targeting Activation of Specific NF-?B Subunits Prevents Stress-Dependent Atherothrombotic Gene Expression

doi: 10.2119/molmed.2012.00282

Figure Lengend Snippet: (A) Suppression of cellular antioxidative defense is dependent on PI3K and cRel. ApoE−/− mice were either untreated or underwent 20 min immobilization in the absence or presence of PKC, p38MAPK or PI3K inhibitors as described. Frozen aortic sections were stained for nitrotyrosine (Magnification: 20×). (B, C) Treatment with antioxidant leads to nonselective inhibition of NF-κB. THP-1 cells were preincubated for 6 h in the absence or presence of pDTC (10 μmol/L), AcCys (5 mmol/L), vitamin E (600 U/mL) or vitamin C (2 mmol/L) before exposure to NE for 15 min or 1 h. (B) Recruitment of the NF-κB subunits p50, p65 and cRel to the NF-κB site of the ICAM-1 promoter, assayed by ChIP, following treatment with antioxidants. (C) RT-PCR for ICAM-1, TF and MnSOD transcription. The experiment was repeated twice with identical results. (D) NF-κB complexes differentially regulate multiple gene products. THP-1 cells were stimulated with 10 nmol/L NE for 1 h after being pretreated with asODN for either p50, cRel or both for 48 h before quantitative RT-PCR profiling arrays for the expression of genes involved in, the antioxidative defense or atherosclerosis were performed. Diagrams are showing the distribution of genes suppressed by respective asODN (See Supplementary Table S5 for complete list of the respective genes).

Article Snippet: Membranes were incubated with anti-NF-κB p50 (Epitomics, Burlingame, CA, USA), anti-NF-κB p65 and anti-NF-κB cRel (Rockland Im-munochemicals, Gilbertsville, PA, USA) antibodies (1:500 dilution).

Techniques: Staining, Inhibition, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Expressing