Journal: Journal of Endocrinology

Article Title: Role of glucocorticoid receptor and CCAAT/enhancer-binding protein α in the feed-forward induction of 11β-hydroxysteroid dehydrogenase type 1 expression by cortisol in human amnion fibroblasts

doi: 10.1677/joe-07-0303

Figure Lengend Snippet: Figure 1 Illustration of the positions and sequences of primers used for sub-cloning 11b-HSD1 promoter. All the sub-cloned promoters carried KpnI and XhoI sites and were cloned into the corresponding sites upstream to luciferase (Luc) reporter gene of pGL3 enhancer plasmid. Boxed nucleotides are putative glucocorticoid- response element and C/EBP-binding site and the underlined nucleotides were mutated for both reporter gene study and EMSA study.

Article Snippet: All the above subcloned sequences and mutation sites were verified by examining the size of PCR products upon gel electrophoresis as well as by sequencing. www.endocrinology-journals.org Transient transfection of WISH cells with pGL3 enhancer plasmid carrying 11b-HSD1 promoter-driven luciferase reporter gene and measurement of promoter activity Amnion epithelium-derived WISH cells were obtained from the American Type Culture Collection (Rockville, MD, USA) with the intention of overcoming relatively poor transfection efficiency in primary cells.

Techniques: Subcloning, Clone Assay, Luciferase, Plasmid Preparation, Binding Assay

Journal: Journal of Endocrinology

Article Title: Role of glucocorticoid receptor and CCAAT/enhancer-binding protein α in the feed-forward induction of 11β-hydroxysteroid dehydrogenase type 1 expression by cortisol in human amnion fibroblasts

doi: 10.1677/joe-07-0303

Figure Lengend Snippet: Figure 4 Top panel: PCR and western blotting analysis showing glucocorticoid receptor (GR) mRNA and protein expression in WISH cells (nZ4). Bottom panel: The basal (top panel, nZ4–12) and cortisol ((1 mM)- induced (bottom panel, nZ4–12) 11b-HSD1 promoter activity in WISH cells. *P!0.05, **P!0.01, comparison between basal activities of various lengths of promoters; #P!0.05, comparison between cortisol- induced activity with respective basal activity. pGL3e: empty pGL3 enhancer plasmid.

Article Snippet: All the above subcloned sequences and mutation sites were verified by examining the size of PCR products upon gel electrophoresis as well as by sequencing. www.endocrinology-journals.org Transient transfection of WISH cells with pGL3 enhancer plasmid carrying 11b-HSD1 promoter-driven luciferase reporter gene and measurement of promoter activity Amnion epithelium-derived WISH cells were obtained from the American Type Culture Collection (Rockville, MD, USA) with the intention of overcoming relatively poor transfection efficiency in primary cells.

Techniques: Western Blot, Expressing, Activity Assay, Comparison, Plasmid Preparation

Journal: Cell Death & Disease

Article Title: TonEBP modulates the protective effect of taurine in ischemia-induced cytotoxicity in cardiomyocytes

doi: 10.1038/cddis.2015.372

Figure Lengend Snippet: Taurine could promote the transcriptional activity of TauT by TonEBP-TonE occupancy in the TauT promoter region. ( a ) Analysis of TauT promoter showed a conserved TonE-binding sites at −110 bases and −109 bases from the transcription start site. ( b ) In the ChIP assay, the TonEBP-TonE occupancy decreased significantly under ischemic insults, and taurine could increase the binding of TonEBP with TonE significantly both in vivo and in vitro ( N =6). ( c ) The proximal site of TauT promoter region (−99 to −124 bp) functions as an efficient cis -element of TonEBP, in the ischemia and taurine-modulated regulation of TauT ( N =4). The luciferase activities in cells transfected with pTauT/-124-Luc decreased significantly under ischemia, whereas taurine strongly attenuated such reduction. In the cells transfected by the pTauT/-99-Luc or pTauT/-124mut-Luc reporter, the luciferase activities decreased to an extremely low level. The luciferase activities (data were expressed as mean±S.E.M. ** P <0.01; ## P <0.01 versus the OGD group in c ; NS, no significance versus the control group; CTL, control group)

Article Snippet: In this part, we obtained the human TauT promoter-driven luciferase reporter plasmid pGL3 (Promega) by using a 1100-bp fragment of the TauT promoter region DNA as the templates for PCR.

Techniques: Activity Assay, Binding Assay, In Vivo, In Vitro, Luciferase, Transfection, Control

Journal: bioRxiv

Article Title: Birth of new protein folds and functions in the virome

doi: 10.1101/2024.01.22.576744

Figure Lengend Snippet: ( A ) Some innate immune pathways in eukaryotes and prokaryotes rely on a viral synthase sensor that detects virus-associated molecular patterns such as dsDNA or dsRNA and generates a nucleotide second messenger that stimulates an antiviral effector. This includes the human cGAS and OAS pathways, as well as prokaryotic CBASS pathways. ( B ) A phylogenetic tree showing the polyphyletic lineages of ligT-like PDEs. A structural search was used to identify viral proteins with a conserved ligT-like fold, and close viral and non-viral members of each were used to construct a phylogenetic tree. Shaded boxes indicate a lineage, with red nodes indicating viral proteins and black nodes indicating non-viral proteins. The red residues in each protein structure are the conserved catalytic histidines. ( C ) A sequence logo showing conservation of the catalytic histidines in viral ligT-like PDEs despite exceptionally low sequence identity overall. ( D ) Outline of the experimental system to assess degradation of 2’3’ cGAMP by viral ligT-like PDEs. HEK 293T cells were transfected with constructs encoding STING, firefly luciferase driven by an IFN-beta promoter, a constitutive renilla luciferase, and a transgene. After 5 hours, cells were treated with 10ug/mL 2’3’ cGAMP or 0.1uM diABZI (a non-cyclic dinucleotide STING agonist). Around 24 hours after the first transfection, luminescence of the firefly and renilla luciferases were measured. ( E ) LigT-like PDEs from avian poxviruses are active against 2’3’ cGAMP. X axis indicates the relative RLU, with all diABZI-treated conditions being normalized to the diABZI RLU of cells transfected with a noncoding transgene and all 2’3’ cGAMP-treated conditions being normalized to the 2’3’ cGAMP RLU of cells transfected with a noncoding transgene. RLUs were initially normalized as (firefly RLU)/(renilla RLU). The Y axis indicates the transgene, with the color of each box indicating the treatment (cGAMP or diABZI). “Mut” indicates the transgene contains H→A mutations of both catalytic histidines.

Article Snippet: The day after plating, cells was transfected with 15ng STING (pMSCV-hygro-STING R232, Addgene 102608), 20ng encoding firefly luciferase driven by an IFN-beta promoter (IFN-Beta_pGL3, Addgene 102597), 5ng of renilla luciferase (pRL-TK - Promega E2241), and 20ng of each putative PDE using the Mirus TransITX2 transfection reagent.

Techniques: Virus, Construct, Sequencing, Transfection, Luciferase

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Transmembrane G protein-coupled receptor 5 signaling stimulates fibroblast growth factor 21 expression concomitant with up-regulation of the transcription factor nuclear receptor Nr4a1.

doi: 10.1016/j.biopha.2021.112078

Figure Lengend Snippet: Fig. 1. Effects of 3-O-acetyloleanolic acid isolated from Forsythiae Fructus on the secretion of FGF21 in C2C12 myotubes. (A) Outline of the extraction and isolation of 3-O-acetyloleanolic acid from Forsythiae Fructus. (B) Structure of 3-O-acetyloleanolic acid. (C) The secretion of FGF21 was increased by 3-O-acetyloleanolic acid isolated from Forsythiae Fructus in C2C12 myotubes. Each value is the mean ± SEM of four separate experiments. The double asterisk indicates P < 0.01.

Article Snippet: The Fgf21 promoter-driven luciferase reporter construct, mFgf21pro1-luc, containing a DNA fragment corresponding to − 1326 to +100 in the region upstream of the murine Fgf21 transcription initiation site linked to the luciferase reporter gene, was a gift from Seiichi Oyadomari (Addgene plasmid #101797; http://n2t.net/addgene:101797; RRID: Addgene_101797) [40].

Techniques: Isolation, Extraction

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Transmembrane G protein-coupled receptor 5 signaling stimulates fibroblast growth factor 21 expression concomitant with up-regulation of the transcription factor nuclear receptor Nr4a1.

doi: 10.1016/j.biopha.2021.112078

Figure Lengend Snippet: Fig. 2. 3-O-Acetyloleanolic acid up- regulates FGF21 expression in C2C12 myotubes. (A) The secretion of FGF21 was increased by 3-O-acetyloleanolic acid in a concentration-dependent manner in C2C12 myotubes. Cells were treated with the indicated concentration of 3-O-acetyloleanolic acid for 20 h, then the media were collected and pro cessed for ELISA. Each value represents the mean ± SEM of five separate ex periments. **P < 0.01, comparison with vehicle control. (B) FGF21 mRNA expression was up-regulated by 3-O- acetyloleanolic acid in C2C12 myo tubes. Cells were treated with 30 µM 3- O-acetyloleanolic acid for 2 and 5 h, harvested, and then processed for real- time qRT-PCR. Each value represents the mean ± SEM of five separate ex periments. *P < 0.01; **P < 0.01, com parison with vehicle control.

Article Snippet: The Fgf21 promoter-driven luciferase reporter construct, mFgf21pro1-luc, containing a DNA fragment corresponding to − 1326 to +100 in the region upstream of the murine Fgf21 transcription initiation site linked to the luciferase reporter gene, was a gift from Seiichi Oyadomari (Addgene plasmid #101797; http://n2t.net/addgene:101797; RRID: Addgene_101797) [40].

Techniques: Expressing, Concentration Assay, Enzyme-linked Immunosorbent Assay, Comparison, Control, Quantitative RT-PCR

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Transmembrane G protein-coupled receptor 5 signaling stimulates fibroblast growth factor 21 expression concomitant with up-regulation of the transcription factor nuclear receptor Nr4a1.

doi: 10.1016/j.biopha.2021.112078

Figure Lengend Snippet: Fig. 3. 3-O-Acetyloleanolic acid stimulates FGF21 expression in mice. (A) Effect of the intraperitoneal administration of 3-O-acetyloleanolic acid at 30 mg/kg on FGF21 mRNA levels in soleus muscle. Total RNA was isolated from the soleus muscle of saline-treated and 3-O-acetyloleanolic acid-treated mice. FGF21 mRNA was determined by qRT-PCR using specific primers. Relative mRNA levels were normalized to 18S. Each value is the mean ± SEM of five mice. The single asterisk indicates P < 0.05. (B) 3-O-acetyloleanolic acid at 30 mg/kg increased circulating FGF21. The plasma FGF21 concentration was determined by FGF21-specific ELISA after the intraperitoneal administration of 3-O-acetyloleanolic acid at 30 mg/kg. Each value represents the mean ± SEM of five mice. The double asterisk in dicates P < 0.01.

Article Snippet: The Fgf21 promoter-driven luciferase reporter construct, mFgf21pro1-luc, containing a DNA fragment corresponding to − 1326 to +100 in the region upstream of the murine Fgf21 transcription initiation site linked to the luciferase reporter gene, was a gift from Seiichi Oyadomari (Addgene plasmid #101797; http://n2t.net/addgene:101797; RRID: Addgene_101797) [40].

Techniques: Expressing, Isolation, Saline, Quantitative RT-PCR, Clinical Proteomics, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Transmembrane G protein-coupled receptor 5 signaling stimulates fibroblast growth factor 21 expression concomitant with up-regulation of the transcription factor nuclear receptor Nr4a1.

doi: 10.1016/j.biopha.2021.112078

Figure Lengend Snippet: Fig. 5. Role of p38 MAPK in the up-regulation of FGF21 secretion caused by 3-O-acetyloleanolic acid in C2C12 cells. (A) Cells were treated with 30 µM 3-O-ace tyloleanolic acid for 10 min, harvested, and then processed for western blotting analysis of the phosphorylated and total p38 levels in whole cell lysate. Western blots were quantified using ImageJ software (National Institutes of Health). Each value represents the mean ± SEM of four to five separate experiments. **P < 0.01, comparison with vehicle control. (B) Relative luciferase activity of the FGF21 promoter construct (mFgf21pro1-luc) in C2C12 cells following treatment with 3-O- acetyloleanolic acid and co-transfection of the TGR5-expression plasmid. The total amount of DNA transfected was standardized with an empty vector. After transfection, the cells were preincubated with the indicated concentrations of SB203580 for 20 min and then stimulated with 30 μM 3-O-acetyloleanolic acid for 8 h. Luciferase activity was normalized by β-gal and expressed relative to that in vehicle-treated cells. Each value is the mean ± SEM of five independent experiments. **P < 0.01, comparison of 3-O-acetyloleanolic acid concentrations. ##P < 0.01, comparison of SB203580 concentrations. (C) Cells were incubated for 20 h with 3-O- acetyloleanolic acid and SB203580, and FGF21 production was determined by ELISA. Each value represents the mean ± SEM of five separate experiments. **P < 0.01, comparison of 3-O-acetyloleanolic acid concentrations. ##P < 0.01, comparison of SB203580 concentrations. (D) Cells were incubated for 5 h in 3-O- acetyloleanolic acid and SB203580, and Fgf21 mRNA levels were determined by qRT-PCR. Fgf21 mRNA levels were normalized relative to 18S mRNA levels. Each value represents the mean ± SEM of five experiments. **P < 0.01, comparison of 3-O-acetyloleanolic acid concentrations. ##P < 0.01, comparison of SB203580 concentrations.

Article Snippet: The Fgf21 promoter-driven luciferase reporter construct, mFgf21pro1-luc, containing a DNA fragment corresponding to − 1326 to +100 in the region upstream of the murine Fgf21 transcription initiation site linked to the luciferase reporter gene, was a gift from Seiichi Oyadomari (Addgene plasmid #101797; http://n2t.net/addgene:101797; RRID: Addgene_101797) [40].

Techniques: Western Blot, Software, Comparison, Control, Luciferase, Activity Assay, Construct, Cotransfection, Expressing, Plasmid Preparation, Transfection, Incubation, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Transmembrane G protein-coupled receptor 5 signaling stimulates fibroblast growth factor 21 expression concomitant with up-regulation of the transcription factor nuclear receptor Nr4a1.

doi: 10.1016/j.biopha.2021.112078

Figure Lengend Snippet: Fig. 6. 3-O-acetyloleanolic acid up-regulates FGF21 expression concomitant with the up-regulation of Nr4a1 in C2C12 myotubes. (A) Nr4a1 mRNA expression was up-regulated by 3-O-acetyloleanolic acid in C2C12 myotubes. Cells were treated with 30 µM 3-O-acetyloleanolic acid for 1, 2 and 5 h, harvested, and then processed for real-time qRT-PCR. Each value represents the mean ± SEM of four separate experiments. *P < 0.05, comparison with vehicle control. (B) Cells were incubated for 5 h with 3-O-acetyloleanolic acid and SB203580, and Nr4a1 mRNA levels were determined by qRT-PCR. Nr4a1 mRNA levels were normalized relative to 18S mRNA levels. Each value represents the mean ± SEM of four separate experiments. *P < 0.05, comparison of 3-O-acetyloleanolic acid concentrations. ##P < 0.01, com parison of SB203580 concentrations. (C) Cells were transfected with a wild-type FGF21 promoter construct (mFgf21pro1-luc) or NBRE-mutated version (NBRE-mut- mFgf21pro1-luc), and relative luciferase activity was measured. Data are means ± SEM of four separate experiments. **P < 0.01, comparison of 3-O-acetyloleanolic acid concentrations. ##P < 0.01, comparison of constructs.

Article Snippet: The Fgf21 promoter-driven luciferase reporter construct, mFgf21pro1-luc, containing a DNA fragment corresponding to − 1326 to +100 in the region upstream of the murine Fgf21 transcription initiation site linked to the luciferase reporter gene, was a gift from Seiichi Oyadomari (Addgene plasmid #101797; http://n2t.net/addgene:101797; RRID: Addgene_101797) [40].

Techniques: Expressing, Quantitative RT-PCR, Comparison, Control, Incubation, Transfection, Construct, Luciferase, Activity Assay