Journal: Scientific Reports

Article Title: A new p65 isoform that bind the glucocorticoid hormone and is expressed in inflammation liver diseases and COVID-19

doi: 10.1038/s41598-021-02119-z

Figure Lengend Snippet: Organization of the murine and human relA gene, homology between amino acid sequences of human and mouse isoforms. ( a ) Partial genomic organization of the murine (GenBank accession number MN508964) and human (GenBank accession number MN508965) relA gene showing the alternatively spliced exon − 1. The lengths of the introns are indicated in base pairs and p65 iso5 start codon is indicated in bold. ( b ) Comparison of mouse and human exon − 1 partial sequences. ( c ) Alignment between amino acid sequences of p65 iso3 mouse and p65 iso5 human. The alignment was performed by using Clustal Omega tool (EMBL-EBI). ( d ) Exon–intron borders of part of the murine and the human relA gene. The intronic splice donor and acceptor sites are underlined. ( e ) Sequences surrounding p65 and p65 iso5 translation start site in mouse and human mRNA. The homology between Kozak consensus sequence of p65 and p65 iso5 are underlined. R indicate a purine (A or G). ( f ) Primary amino acid sequences of human p65 and p65 iso5 proteins. Underlined: NF-κB/Rel domain signature. Bold: Rel Homology Domain starts at amino acid 19. Italics: first methionine for either p65 or p65 iso5.

Article Snippet: Finished the run, the proteins were transferred to nitrocellulose membrane (Amersham Protran 0.45 NC nitrocellulose Western blotting membranes GE Healthcare) overnight at 4 °C, in Tris/glycine/methanol 20% buffer at 50 V. The membranes were then blocked for 30′ in a milk solution (5% dry powdered non-fat milk, PBS 1×, Tween 20 0,1%, with 100 μl/l of protease inhibitor cocktail) at room temperature and incubated for 2 h in a milk solution, with NF-kB p65 (G-8) (sc-398442) or NF-kB p65 (c-20) (sc-372) antibody dilution 1:5000 or NF-kB p65 (A) antibody (sc-109) (Santa Cruz Biotechnology) dilution 1:5000.

Techniques: Comparison, Sequencing

Journal: Scientific Reports

Article Title: A new p65 isoform that bind the glucocorticoid hormone and is expressed in inflammation liver diseases and COVID-19

doi: 10.1038/s41598-021-02119-z

Figure Lengend Snippet: Nested RT-PCR analysis of p65 iso5 expression in RNA samples from human and mouse brain in different mouse tissues. ( a ) First round PCR reaction was performed with specific oligonucleotides for the exon − 1 and 3′UTR (untranslated region). For human and mouse RNA, subsequent RT-PCR reactions were performed with oligonucleotide specific for exon-1 and exon 5; exon 7; exon 9; exon 10 (the entire transcript). PCR analysis of mouse brain RNA were performed with specific primers for: exon − 1 and exon 2; exon − 1 and exon 0; exon 0 and exon 2. The + and − symbols indicate the presence and the absence respectively of reverse transcriptase in the reaction. ( b ) Nested RT-PCR performed on mouse RNA samples extracted from different tissues. First round PCR was performed with primers located on exon − 1 and 3′UTR region, second round PCR was performed with oligonucleotides specific for exon 2 and 10. The − symbol indicates the absence of reverse transcriptase in the reaction (representative of each sample). The full length images of the gels are available in the “ ” file.

Article Snippet: Finished the run, the proteins were transferred to nitrocellulose membrane (Amersham Protran 0.45 NC nitrocellulose Western blotting membranes GE Healthcare) overnight at 4 °C, in Tris/glycine/methanol 20% buffer at 50 V. The membranes were then blocked for 30′ in a milk solution (5% dry powdered non-fat milk, PBS 1×, Tween 20 0,1%, with 100 μl/l of protease inhibitor cocktail) at room temperature and incubated for 2 h in a milk solution, with NF-kB p65 (G-8) (sc-398442) or NF-kB p65 (c-20) (sc-372) antibody dilution 1:5000 or NF-kB p65 (A) antibody (sc-109) (Santa Cruz Biotechnology) dilution 1:5000.

Techniques: Reverse Transcription Polymerase Chain Reaction, Expressing, Reverse Transcription

Journal: Scientific Reports

Article Title: A new p65 isoform that bind the glucocorticoid hormone and is expressed in inflammation liver diseases and COVID-19

doi: 10.1038/s41598-021-02119-z

Figure Lengend Snippet: Biochemical and functional analysis of p65 iso5 protein and localization in the cell. ( a ) Electro mobility shift assay (EMSA) showing p65 and p65 iso5 binding activity to the radiolabelled NF-κB consensus sequence. The specific bands were suppressed by 100-fold excess of unlabelled probe. The full length image of the gel is available in the “ ” file. ( b ) Polyacrylamide gel electrophoresis showing the in vitro transcribed and translated radiolabelled p65 and p65 iso5 proteins. The membrane has been cut prior the exposition. ( c ) Analysis of the transcriptional activity of p65 and p65 iso5. HeLa cells were transfected with a luciferase reporter driven by consensus NF-κB response elements and with the indicated expression vector. ( d ) Opposite effects of p65 iso5 and p65 on the transcriptional activity of the glucocorticoid receptor (GR) induced by dexamethasone. HeLa cells were transfected with luciferase reporter driven by specific glucocorticoid response elements (GRE) and the indicated plasmids. After transfection, GR was activated by the administration of the glucocorticoid agonist dexamethasone. ( e ) Representative of immunofluorescence of Cos-1 cells transfected with p65 and p65 iso5. In red we identified the protein using NF-κB p65 (L8F6) PE conjugate antibody that recognizes C-terminal proteins, nuclei are evidenced in blue. ( f ) Localization of p65 and p65 iso5 fused at 5′ with GFP (green fluorescent protein), nuclei are evidenced in blue. Data information: In ( b , c ), data are presented as mean ± SEM. *P < 0.001, in comparison to cells transfected with reporter plasmid. (GRE) and the indicated plasmids. After transfection, GR was activated by the administration of the glucocorticoid agonist dexamethasone. The full length images of EMSA and of the in vitro transcribed and translated radiolabelled p65 and p65 iso5 proteins are available in the “ ” file.

Article Snippet: Finished the run, the proteins were transferred to nitrocellulose membrane (Amersham Protran 0.45 NC nitrocellulose Western blotting membranes GE Healthcare) overnight at 4 °C, in Tris/glycine/methanol 20% buffer at 50 V. The membranes were then blocked for 30′ in a milk solution (5% dry powdered non-fat milk, PBS 1×, Tween 20 0,1%, with 100 μl/l of protease inhibitor cocktail) at room temperature and incubated for 2 h in a milk solution, with NF-kB p65 (G-8) (sc-398442) or NF-kB p65 (c-20) (sc-372) antibody dilution 1:5000 or NF-kB p65 (A) antibody (sc-109) (Santa Cruz Biotechnology) dilution 1:5000.

Techniques: Functional Assay, Electro Mobility Shift Assay, Binding Assay, Activity Assay, Sequencing, Polyacrylamide Gel Electrophoresis, In Vitro, Membrane, Transfection, Luciferase, Expressing, Plasmid Preparation, Immunofluorescence, Comparison

Journal: Scientific Reports

Article Title: A new p65 isoform that bind the glucocorticoid hormone and is expressed in inflammation liver diseases and COVID-19

doi: 10.1038/s41598-021-02119-z

Figure Lengend Snippet: Transcriptional regulation of p65 iso5 on human IL-6-luc and TNFα-luc promoters. ( a ) Effect of p65 iso5 on natural human promoter IL-6-luc. HeLa cells were cotransfected with a luciferase reporter driven by promoter IL6-651luc; IL6-651luc mut κB; IL6-651luc mut GRE; IL-6-651luc mut AP-1; IL-6-651luc mut CRE; IL-6-651luc mut c/EBP and the plasmids p50, p65 and p65 iso5. ( b ) HeLa cells were cotransfected with a luciferase reporter driven by promoter TNFα-luc and the indicated plasmids. Cells were maintained in RPMI 1640 supplemented with Charcoal–dextran (CD) Fetal Bovine Serum (FBS). Data information: in ( a , b ), data are presented as mean ± SEM. *P < 0.001, in comparison to cells transfected with reporter plasmid.

Article Snippet: Finished the run, the proteins were transferred to nitrocellulose membrane (Amersham Protran 0.45 NC nitrocellulose Western blotting membranes GE Healthcare) overnight at 4 °C, in Tris/glycine/methanol 20% buffer at 50 V. The membranes were then blocked for 30′ in a milk solution (5% dry powdered non-fat milk, PBS 1×, Tween 20 0,1%, with 100 μl/l of protease inhibitor cocktail) at room temperature and incubated for 2 h in a milk solution, with NF-kB p65 (G-8) (sc-398442) or NF-kB p65 (c-20) (sc-372) antibody dilution 1:5000 or NF-kB p65 (A) antibody (sc-109) (Santa Cruz Biotechnology) dilution 1:5000.

Techniques: Luciferase, Comparison, Transfection, Plasmid Preparation

Journal: Scientific Reports

Article Title: A new p65 isoform that bind the glucocorticoid hormone and is expressed in inflammation liver diseases and COVID-19

doi: 10.1038/s41598-021-02119-z

Figure Lengend Snippet: Modelling and dexamethasone binding of p65 iso5. ( a ) The p65 iso5 ipotetical modelling was determined with Modeller v9.8. ( b ) The p65 crystallized structure obtained from the complex IκBα/NF-κB (PDB: 1NFI). ( c , d ) The docking simulations of the p65 and p65 iso5 were performed with Autodock v4.2 program. In the red box is highlighted the β-sheet element of the first 31 amino acids of the wild-type protein while in the p65 iso5 the 31 amino acids are missing and create a free pocket for the dexamethasone binding. (e ) The Bioluminescence Resonance Energy Transfer (BRET) assay shows the in vivo interaction between the p65 iso5 and dexamethasone. Cos-1 cells were cotransfected with the BRET constructs and treated with Dex-FITC, the raw BRET ratio was calculated by the A/D (λ 530 nm acceptor/λ 495 nm donor). Data information: in ( f ), data are presented as mean ± SEM. *P < 0.001, in comparison to cells transfected without Dex-FITC.

Article Snippet: Finished the run, the proteins were transferred to nitrocellulose membrane (Amersham Protran 0.45 NC nitrocellulose Western blotting membranes GE Healthcare) overnight at 4 °C, in Tris/glycine/methanol 20% buffer at 50 V. The membranes were then blocked for 30′ in a milk solution (5% dry powdered non-fat milk, PBS 1×, Tween 20 0,1%, with 100 μl/l of protease inhibitor cocktail) at room temperature and incubated for 2 h in a milk solution, with NF-kB p65 (G-8) (sc-398442) or NF-kB p65 (c-20) (sc-372) antibody dilution 1:5000 or NF-kB p65 (A) antibody (sc-109) (Santa Cruz Biotechnology) dilution 1:5000.

Techniques: Binding Assay, Bioluminescence Resonance Energy Transfer, In Vivo, Construct, Comparison, Transfection

Journal: Scientific Reports

Article Title: A new p65 isoform that bind the glucocorticoid hormone and is expressed in inflammation liver diseases and COVID-19

doi: 10.1038/s41598-021-02119-z

Figure Lengend Snippet: mRNA relative quantity of p65 and p65 iso5 and level IL-6 in blood. ( a ) The Ct and median measuring gene expression levels of p65 and p65 iso5 in PBMC of SARS-CoV-2 and in control patients. The p65 and p65 iso5 mRNA are significantly up-regulated in patients with SARS-CoV-2 compared to control samples. ( b ) IL-6 levels and median measured in patients with SARS-CoV-2 compared to healthy patients. In ( a , b ) data are presented as mean ± SEM. ****< 0.001 and ***< 0.005 in comparison to control patients.

Article Snippet: Finished the run, the proteins were transferred to nitrocellulose membrane (Amersham Protran 0.45 NC nitrocellulose Western blotting membranes GE Healthcare) overnight at 4 °C, in Tris/glycine/methanol 20% buffer at 50 V. The membranes were then blocked for 30′ in a milk solution (5% dry powdered non-fat milk, PBS 1×, Tween 20 0,1%, with 100 μl/l of protease inhibitor cocktail) at room temperature and incubated for 2 h in a milk solution, with NF-kB p65 (G-8) (sc-398442) or NF-kB p65 (c-20) (sc-372) antibody dilution 1:5000 or NF-kB p65 (A) antibody (sc-109) (Santa Cruz Biotechnology) dilution 1:5000.

Techniques: Gene Expression, Control, Comparison

Journal: Scientific Reports

Article Title: A new p65 isoform that bind the glucocorticoid hormone and is expressed in inflammation liver diseases and COVID-19

doi: 10.1038/s41598-021-02119-z

Figure Lengend Snippet: p65 and p65 iso5 protein expression by 2-DE in human liver samples, transfected cell lines and human liver carcinoma cells. ( a ) Expression of p65 and p65 iso5 proteins by 2-DE in Cos-1 and HeLa cells transfected with p65 and p65 iso5 proteins with specific antibodies for C-terminal and N-terminal epitope region. (b ) Expression of p65 and p65 iso5 proteins by 2-DE in HepG2 and HUH7 hepatocarcinoma cell lines. (c ) Electrophoretic pattern of the p65 and p65 iso5 proteins on human liver of healthy, cirrhosis, and hepatocellular carcinoma (HCC) samples. The p65 antibody is specific for an epitope at C-terminal region that is present both in p65 and in p65 iso5 proteins. (d ) Theoretical Molecular weight (Mw) and Isoelectric point (pI) of p65 and p65 iso5. The values were calculated using Compute pI/Mw tool (ExPASy) and do not take into consideration the possible post-translational modifications (PTMs). Schematic representation based on the hypothetical pI and Mw of the p65 and p65 iso5 protein pattern on 2D-PAGE. The full length images of 2-DE are available in the “ ” file.

Article Snippet: Finished the run, the proteins were transferred to nitrocellulose membrane (Amersham Protran 0.45 NC nitrocellulose Western blotting membranes GE Healthcare) overnight at 4 °C, in Tris/glycine/methanol 20% buffer at 50 V. The membranes were then blocked for 30′ in a milk solution (5% dry powdered non-fat milk, PBS 1×, Tween 20 0,1%, with 100 μl/l of protease inhibitor cocktail) at room temperature and incubated for 2 h in a milk solution, with NF-kB p65 (G-8) (sc-398442) or NF-kB p65 (c-20) (sc-372) antibody dilution 1:5000 or NF-kB p65 (A) antibody (sc-109) (Santa Cruz Biotechnology) dilution 1:5000.

Techniques: Expressing, Transfection, Molecular Weight

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Protective role of hydrogen sulfide against diabetic cardiomyopathy by inhibiting pyroptosis and myocardial fibrosis.

doi: 10.1016/j.biopha.2024.116613

Figure Lengend Snippet: Fig. 5. High glucose induced cardiomyocyte pyroptosis.A.Immunofluorescence was used to identify the purity of NRVMs.B.Calcein-AM/Hoechst 33342 staining was used to observe cardiomyocyte membrane injury caused by high glucose.C.Scanning electron microscopy was used to observe the effect of high glucose on NRVMs damage.D-G.High glucose induced increased expressions of pyroptosis related proteins in NRVMs.Values are expressed as mean±SD (n=4).*P<0.05,**P<0.01vs Control group.

Article Snippet: Initially, a 2 μM Calcein AM staining solution (Beyotime, China) was formulated using a serum-free cell culture medium.

Techniques: Immunofluorescence, Staining, Membrane, Electron Microscopy, Control