p65 gfp rela  (Addgene inc)

Journal: Biochimica et biophysica acta. Molecular and cell biology of lipids

Article Title: Potential of omega-3 and conjugated fatty acids to control microglia inflammatory imbalance elicited by obesogenic nutrients.

doi: 10.1016/j.bbalip.2023.159331

Figure Lengend Snippet: Fig. 5. Omega-3, CLNA and CLA fatty acids can revert Fructose and Palmitic acid-induced microglia inflammatory imbalance elicited by NF-κB pathway activation, ROS production and Src Tyrosine activation. (A) Fluorescence imaging and results of the quantification of human microglia cell line (HMC3) expressing the miRFP703-Ikβα sensor in cells subjected to the stimulus solution (Palmitic acid+Fructose) and in cells pre-incubated with the studied fatty acids (omega-3, CLA and CLNA) for the selected time points (0, 5, 10 and 15 min). A decreased signal means a bigger Ikβα degradation and higher NF-κB pathway activation. Error bar represents the SEM calculated from n > 10 cells from two independent cultures. Two-way ANOVA in relation to Palmitic acid+Fructose (PA + Frut), where no pre-incubation with the selected fatty acids was performed. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (B) Fluorescence imaging and results of the quantification of human microglia cell line (HMC3) expressing the GFP-p65 and mneptune biosensor in cells subjected to the stimulus solution (Palmitic acid+Fructose) and in cells pre-incubated with the studied fatty acids (omega-3, CLA and CLNA). The mnpetune probe allows nucleus staining. The results correspond to n of the sensor signal of human microglia expressing the GFP-p65 sensor after the 15 min incubation time. This variation is in relation to the baseline measured for each experiment. A negative signal means a decreased signal in relation to the baseline, meaning that the catalytic p65 subunit of the NF-κB complex migration to the nucleus decreased in cells exposed to a pre-incubation with the studied fatty acids. In cells only subjected to the stimulus solution the signal increased after baseline measure, indicating catalytic p65 subunit translocation to the nucleus. Error bar represents the SEM calculated from n > 10 cells from two independent cultures. One-way ANOVA in relation to Palmitic acid+Fructose (PA + Frut), where no pre-incubation with the selected fatty acids was performed. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (C) Fluorescence imaging and results of the quantification of human microglia cell line (HMC3) expressing the ROS FRET sensor HSP in cells subjected to the stimulus solution (Palmitic acid+Fructose) and in cells pre-incubated with the studied fatty acids (omega-3, CLA and CLNA) for the selected time points (0, 5, 10 and 15 min). Error bar represents the SEM calculated from n > 15 cells from two independent cultures. Two-way ANOVA in relation to Palmitic acid+Fructose (PA + Frut), where no pre-incubation with the selected fatty acids was performed. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (D) Fluorescence imaging and quantification of human microglia cell line (HMC3) expressing the LynSrc FRET sensor in cells subjected to the stimulus solution (Palmitic acid+Fructose) and in cells pre-incubated with the studied fatty acids (omega-3, CLA and CLNA) for the selected time points (0, 5, 10 and 15 min). Error bar represents the SEM calculated from n > 15 cells from two independent cultures. Two-way ANOVA in relation to Palmitic acid+Fructose (PA + Frut), where no pre- incubation with the selected fatty acids was performed. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The measurement of the nuclear accumulation of the p65 subunit of NF-κB as a functional indicator of NF-κB activation was achieved by using the NF-κB GFP-tagged p65 (here defined and GFP-p65; addgene plasmid #23255) [82].

Techniques: Activation Assay, Fluorescence, Imaging, Expressing, Incubation, Staining, Migration, Translocation Assay

Journal: Science translational medicine

Article Title: Hepatocyte TLR4 triggers inter-hepatocyte Jagged1/Notch signaling to determine NASH-induced fibrosis

doi: 10.1126/scitranslmed.abe1692

Figure Lengend Snippet: (A) Serum LPS, (B) liver Tlr4 mRNA, and (C) liver p65 protein expression in WT mice fed chow or NASH diet for 16 weeks (n = 3 to 6 per group). (D) Jag1 promoter luciferase activity in WT primary hepatocytes cotransfected with p65 (n = 4 per group), and (E) ChIP assay for NF-κB occupancy at the Jag1 promoter in livers from mice fed normal chow or NASH diet for 16 weeks (n = 4 per group). (F) Eight- to 10-week-old Tlr4flox/flox mice were transduced with AAV8-Tbg-GFP (Cre−) or AAV8-Tbg-CRE (L-Tlr4) and then fed NASH diet for 16 weeks (n = 12 to 13 per group). (G) Jagged1 protein expression, (H) expression of Notch targets, and (I) markers of HSC activation. (J) Liver Sirius red staining (n = 4 to 9 per group). (K) Control (pLive-con) or Jag1 expression (pLive-Jagged1) vectors were hydrodynamically administered to adult Tlr4flox/flox mice. Two weeks later, all mice were transduced with AAV8-Tbg-CRE and then fed NASH diet for 16 weeks (n = 8 per group). (L) Expression of Jag1 mRNA and (M) markers of HSC activity. (N) Sirius red staining (n = 5 per group). *P < 0.05, **P < 0.01, and ***P < 0.001 as compared to the indicated controls by two-tailed t tests (two groups). All data are presented as means ± SEM.

Article Snippet: The p65 expression construct ( 72 ) was obtained from Addgene (no. 23255).

Techniques: Expressing, Luciferase, Activity Assay, Transduction, Activation Assay, Staining, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Heterozygous RELA mutations cause early-onset systemic lupus erythematosus by hijacking the NF-κB pathway towards transcriptional activation of type-I Interferon genes

doi: 10.1101/2020.04.27.046102

Figure Lengend Snippet: ( a ) Pedigree and Sanger sequencing of cDNA of the family-1 and 2. In family 1 a heterozygous RELA c.256 C>A missense mutation predicted to result in the p.H86N mutated RELA/p65 protein was found in P1 and P2. In family 2 a heterozygous RELA c.985 C>T nonsense mutation predicted to result in the p.R329X truncated RELA/p65 protein was found in P3. ( b ) RELA/p65 protein structure with indicated mutations positions. RHD = REL-Homology-Domain; NLS = Nuclear localization signal; 9AATAD = 9 amino acid transactivation domain. ( c ) Expression of RELA/p65 in activated T-cell blasts of probands and healthy relative controls by Western Blot analysis using antibodies specific for N-terminal (Nterm) and C-terminal (Cterm) domains of RELA/p65. Anti-β-Actin was used as an internal control. ( d ) Immuno-blotting of HEK293T cell lysates co-transfected with constructs expressing N-terminal HA-tagged RELA/p65 and IκB-α with a ratio of 3:1. Anti-HA-RELA/p65, anti-IκB-α and anti-α-tubulin antibodies were used to assess protein expression. ( e ) Immunoblotting of cell lysates from co-transfected HEK293T cells with an IκB-α construct and HA-RELA/p65 constructs performed as indicated. Proteins were immuno-precipitated with anti-HA antibody. Initial lysates and immune-precipitated proteins were subjected to immunoblotting with anti-RELA/p65, anti-IκB-α anti-NF-κB1/p50 and anti-@-tubulin antibodies. A representative experiment is depicted.

Article Snippet: Site directed mutagenesis using GeneARTTM Site-Directed Mutagenesis System (Thermo Fischer Scientific) was performed following manufacture’s instruction on the RELA WT expression constructs pEBB HA RelA (addgene #74892) and GFP-p65 (addgene #23255).

Techniques: Sequencing, Mutagenesis, Expressing, Western Blot, Control, Transfection, Construct

Journal: bioRxiv

Article Title: Heterozygous RELA mutations cause early-onset systemic lupus erythematosus by hijacking the NF-κB pathway towards transcriptional activation of type-I Interferon genes

doi: 10.1101/2020.04.27.046102

Figure Lengend Snippet: ( a ) Immunofluorescence of activated T-cell blasts from P1 (H86N), P3 (R329X) and healthy donor stimulated 30 minutes with TNF-α and analyzed by confocal microscopy using N-terminus (Nterm)-specific anti-RELA/p65 (Red), C-terminus (Cterm)-specific anti-RELA/p65 (Green) antibodies, and DAPI (4’-6-diamidino-2-phenylindole). Top panels: Representative pictures. Bottom Panel: Quantification of the RELA/p65 nuclear localization with the N-terminal-specific antibody and C-terminal-specific antibody. Mann-Whitney Wilcoxon, * P <0.05; mean±SD. ( b ) Immunofluoresence of HacaT and HEK293T cells transfected with the different RELA constructs analyzed by confocal microscopy. Scale bar = 5®m.

Article Snippet: Site directed mutagenesis using GeneARTTM Site-Directed Mutagenesis System (Thermo Fischer Scientific) was performed following manufacture’s instruction on the RELA WT expression constructs pEBB HA RelA (addgene #74892) and GFP-p65 (addgene #23255).

Techniques: Immunofluorescence, Confocal Microscopy, MANN-WHITNEY, Transfection, Construct

Journal: The Journal of Biological Chemistry

Article Title: Sirtuin 1 reduces hyaluronan synthase 2 expression by inhibiting nuclear translocation of NF-κB and expression of the long-noncoding RNA HAS2–AS1

doi: 10.1074/jbc.RA119.011982

Figure Lengend Snippet: SIRT1 activators affect HAS2–AS1 expression and NF-κB nuclear translocation. A, quantitative RT-PCR analyses are shown for HAS2–AS1 expression in AoSMCs treated for 24 h with 0.1 μg/ml TNFα alone or with 1 μ m SRT1720 or 100 μ m RESV. Data are expressed as relative expression of HAS2–AS1 with respect to its control ( CNTR ). Values are reported as mean ± S.E. of three independent experiments performed in duplicates. *, p < 0.05; **, p < 0.01; ***, p < 0.001. B, quantitative RT-PCR analyses are shown for AoSMCs nucleofected with 50 n m scrambled siRNA or HAS2 siRNA and left untreated or treated with 0.1 μg/ml TNFα for 24 h. Data are shown as mean ± S.E. of three independent experiments. *, p < 0.05; **, p < 0.01. C, representative images are shown for AoSMCs nucleofected with 4 μg of pcDNA3-GFP-RelA plasmid and grown on coverslips. Twenty four hours after the transfection, cells were treated as indicated for 24 h, washed in PBS, and observed by confocal microscopy (×63 objective). Bars , 20 μm.

Article Snippet: To study GFP–p65 subcellular localization 4 μg of pcDNA3-GFP-RELA plasmid (23255, Addgene) was nuclefected in AoSMCs.

Techniques: Expressing, Translocation Assay, Quantitative RT-PCR, Control, Plasmid Preparation, Transfection, Confocal Microscopy

Journal: The Journal of Biological Chemistry

Article Title: Sirtuin 1 reduces hyaluronan synthase 2 expression by inhibiting nuclear translocation of NF-κB and expression of the long-noncoding RNA HAS2–AS1

doi: 10.1074/jbc.RA119.011982

Figure Lengend Snippet: NF-κB blockade influences HAS2–AS1 expression. A , confocal microscopy images are shown for AoSMCs seeded on coverslips, nucleofected with 4 μg of pcDNA3-GFP-RelA, and treated with 0.1 μg/ml TNFα alone or in combination with 10 μ m PDTC (×63 objective). Bars , 20 μ m . Quantitative RT-PCR analyses are shown for HAS2–AS1 ( B ) and HAS2 ( C ) expression of AoSMCs treated with 0.1 μg/ml TNFα alone or with 10 μ m PDTC for 24 h. Data are displayed as mean ± S.E. of three independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001; CNTR , control.

Article Snippet: To study GFP–p65 subcellular localization 4 μg of pcDNA3-GFP-RELA plasmid (23255, Addgene) was nuclefected in AoSMCs.

Techniques: Expressing, Confocal Microscopy, Quantitative RT-PCR, Control

Journal: The Journal of Biological Chemistry

Article Title: Sirtuin 1 reduces hyaluronan synthase 2 expression by inhibiting nuclear translocation of NF-κB and expression of the long-noncoding RNA HAS2–AS1

doi: 10.1074/jbc.RA119.011982

Figure Lengend Snippet: Working model for protective effects of SIRT1 via HAS2–AS1 and NF-κB. Stimulation of AoSMCs with TNFα can activate the p65 subunit of NF-κB through acetylation with its consequent translocation into the nucleus and the activation of target genes involved in the inflammatory response. In the nucleus, p65 is able to activate HAS2–AS1 promoter, which induces HAS2 expression and subsequent synthesis of a monocyte adhesive extracellular HA matrix on AoSMCs. The activation of SIRT1 by SRT1720 and RESV initiates the inhibition pathway ( red ) by retaining p65 in the cytoplasm, probably by promoting deacetylation of p65, which prevents p65 nuclear translocation that prevents activation of an HAS2–AS1 promoter. This would inhibit HAS2 mRNA and protein expression, thereby preventing formation of the monocyte adhesive HA matrix, immune cells recruitment, and AoSMC migration, thus protecting AoSMCs from TNFα-induced inflammation.

Article Snippet: To study GFP–p65 subcellular localization 4 μg of pcDNA3-GFP-RELA plasmid (23255, Addgene) was nuclefected in AoSMCs.

Techniques: Translocation Assay, Activation Assay, Expressing, Adhesive, Inhibition, Migration