Journal: Nucleic Acids Research

Article Title: A bio-orthogonal linear ubiquitin probe identifies STAT3 as a direct substrate of OTULIN in glioblastoma

doi: 10.1093/nar/gkad002

Figure Lengend Snippet: The development and evaluation of the NAEK-Ub probe. ( A ) The scheme of the bio-orthogonal NAEK-Ub strategy. GSCs were transduced with a modified pCDH lentivector for integration of the genes encoding PylRS/tRNA CUA expression cassettes and an amber codon-containing ubiquitin (Ub-54TAG) into the host genome. The resultant transgenic cells, GSC-tRNA/pylRS/Ub-54TAG, were then supplemented with NAEK for 24 h, producing NAEK-Ub which readily conjugates to the substrates of linear Ub. The SPAAC reaction between the azides of the NAEK-Ub chain and biotin–DIBO facilitates enrichment and identification of the linear Ub-modified proteins. ( B ) Ub structure labeled with the surface residues (in blue) and the terminal residues (in red) (generated by PyMOL). The accessible surface area and the stable energy of Ub variants with NAEK substitution on each candidate site (Gly10, Lys29, Arg42, Ala46, Arg54 and Asn60) are shown in . ( C ) The expression and pull-down efficiency of Ub variants with NAEK incorporated at different sites (Gly10, Lys29, Arg42, Ala46, Arg54 and Aln60) in PylRS/tRNA CUA -transduced HEK293T cells. Arg54 was finally chosen as the insertion site for NAEK. ( D ) High resolution tandem MS identification of NAEK-Ub peptides after protease digestion. A representative MS/MS fragment spectrum of the characteristic peptide QLEDG U 54 TLSDYNIQR (by trypsin) is shown. The NAEK insertion site was further confirmed by the GluC-digested peptide G U 54 TLSDYNIQRE. U (in red) represents NAEK. ( E ) CD spectra of wild-type ubiquitin (blue curve) and NAEK-Ub (red curve) in 50 mM sodium phosphate buffer (pH 7.4). ( F ) Wild-type Ub and NAEK-Ub were reacted with the E2 enzyme UBE2D3 in the presence of UBE1 and ATP. The conjugation of Ub and NAEK-Ub onto the E2 via thioester bonds in the reactions was analyzed by SDS–PAGE with or without DTT. ( G ) In vitro ubiquitin chain assembly assay of wild-type Ub and NAEK-Ub. Ub or NAEK-Ub was incubated with the linear ubiquitination machinery (composed of UBE1, UBE2D3 and HOIP RBR-LDD ) and recombinant GST–Ub (acceptor ubiquitin) in the presence or absence of ATP. The ladders of polyUb chains are assessed by immunoblotting using anti-Ub antibody (left) and anti-linear Ub antibody (right), respectively. Standard reaction conditions are described in the Materials and Methods. ( H ) The polyUb chains of wild-type Ub and NAEK-Ub were prepared as described in (G). The resultant reactions were incubated with 0.2 μg/ml OTULIN at 37°C for 1 h. Immunoblotting was performed with anti-linear Ub antibody. The corresponding silver-stained gel is shown in .

Article Snippet: Human HEK293T cells purchased from the ATCC, and stat3-null HeLa cells purchased from ABclonal (20170728-02) were grown in Dulbecco's modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS; FND500, excellbio), 100 U/ml penicillin and 100 U/ml streptomycin.

Techniques: Transduction, Modification, Expressing, Ubiquitin Proteomics, Transgenic Assay, Labeling, Generated, Tandem Mass Spectroscopy, Circular Dichroism, Conjugation Assay, SDS Page, In Vitro, Incubation, Recombinant, Western Blot, Staining

Journal: Nucleic Acids Research

Article Title: A bio-orthogonal linear ubiquitin probe identifies STAT3 as a direct substrate of OTULIN in glioblastoma

doi: 10.1093/nar/gkad002

Figure Lengend Snippet: Global profiling of linear Ub substrates in live cells with NAEK-Ub. ( A ) Schematic workflow of NAEK-Ub proteomics. HEK293T cells were transfected with plasmids of the PylRS/tRNA CUA pair and Ub-54TAG, and were cultured in DMEM supplemented with 1 mM NAEK for 24 h. The resultant cells were serum starved overnight and then treated with TNFα (10 ng/μl) for 15 min. Cell lysates were incubated with biotin–DIBO (10 μM) at room temperature for 2 h and then immunoprecipitated with streptavidin beads. The enriched proteins were digested by trypsin and subjected to LC-MS/MS analysis. ( B ) HEK293T cells transduced with PylRS/tRNA CUA /Ub-54TAG were starved overnight and then treated with TNFα for 15 min. Phosphorylated p65 indicates the activation of NF-κB. The streptavidin pull-down fraction was immunoblotted against anti-linear Ub. ( C ) Differential analysis of NAEK-Ub pull-downs (visualized by volcano plot; source data shown in ) in the TNFα-treated group and the control. The previously reported substrate NEMO was labeled in red as a benchmark. Putative substrates selected for further validation were labeled in orange. ( D ) Hierarchical clustering of differential proteins derived from NAEK-Ub pull-downs between the TNFα-treated group and the control group (source data shown in ). ( E ) Pathway analysis and annotation of proteins enriched in the TNFα-treated group by Gene Ontology Biological Processes (GOBP) and Kyoto Encyclopaedia of Genes and Genomes (KEGG). ( F ) Verification of putative linear Ub substrates. The plasmid of each Flag-tagged candidate protein was transfected into HEK293T cells with or without LUBAC components (Myc-HOIP, HA-HOIL-1L and HA-SHARPIN). Immunoprecipitation was performed with anti-Flag M2 beads. Flag-NEMO was used as a system control.

Article Snippet: Human HEK293T cells purchased from the ATCC, and stat3-null HeLa cells purchased from ABclonal (20170728-02) were grown in Dulbecco's modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS; FND500, excellbio), 100 U/ml penicillin and 100 U/ml streptomycin.

Techniques: Transfection, Cell Culture, Incubation, Immunoprecipitation, Liquid Chromatography with Mass Spectroscopy, Transduction, Activation Assay, Control, Labeling, Biomarker Discovery, Derivative Assay, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: A bio-orthogonal linear ubiquitin probe identifies STAT3 as a direct substrate of OTULIN in glioblastoma

doi: 10.1093/nar/gkad002

Figure Lengend Snippet: STAT3 is a direct substrate of linear Ub. ( A ) Workflow of NAEK-Ub proteomics to identify linear Ub-modified proteins in OTULIN-depleted GSCs. ( B ) Immunoblot analysis of NAEK-Ub pull-downs in shOTULIN GSCs and the control. OTULIN depletion leads to an overall increase of linear Ub signals. ( C ) Volcano plot of the quantitative comparison of the NAEK-Ub proteome in shOTULIN GSCs and the controls (source data are shown in ). STAT3 (labeled in orange) is selected for subsequent validation and functional evaluation. ( D ) Immunoprecipitation (IP) analysis of linear ubiquitination of STAT3 in HEK293T cells co-transfected with LUBAC (comprising Myc-HOIP, HA-HOIL-1L and HA-Sharpin). IP was performed with anti-Flag M2 beads. Flag-NEMO was used as a system control. ( E ) IP analysis of linear ubiquitination of STAT3 in HEK293T cells co-transfected with LUBAC, together with either wild-type OTULIN or its catalytically inactive mutant (OTULIN C129S ). STAT3 IP was performed with anti-Flag M2 beads. ( F ) In vitro linear ubiquitination assay of GST–STAT3 with or without OTULIN. Recombinant GST–STAT3 was incubated with the linear ubiquitination machinery (composed of UBE1, UBE2D3, HOIPRBR-LDD and Ub) with or without OTULIN. The reaction was carried out at 37°C for 2 h and analyzed by immunoblotting. ( G ) IP analysis of linear ubiquitination of endogenous STAT3 in undifferentiated GSCs transduced with either lentiviral shOTULIN or shNC. IP of endogenous STAT3 was performed using anti-STAT3 antibody, and was immunoblotted with anti-linear Ub antibody.

Article Snippet: Human HEK293T cells purchased from the ATCC, and stat3-null HeLa cells purchased from ABclonal (20170728-02) were grown in Dulbecco's modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS; FND500, excellbio), 100 U/ml penicillin and 100 U/ml streptomycin.

Techniques: Modification, Western Blot, Control, Comparison, Labeling, Biomarker Discovery, Functional Assay, Immunoprecipitation, Ubiquitin Proteomics, Transfection, Mutagenesis, In Vitro, Recombinant, Incubation, Transduction

Journal: Nucleic Acids Research

Article Title: A bio-orthogonal linear ubiquitin probe identifies STAT3 as a direct substrate of OTULIN in glioblastoma

doi: 10.1093/nar/gkad002

Figure Lengend Snippet: Identification of linear Ub sites on STAT3. ( A ) The identified linear ubiquitination sites of STAT3 (Lys153, Lys161, Lys163 and Lys191) and their evolutionary conservation in vertebrates. K refers to a lysine residue. ( B ) Representative MS/MS spectra of linear ubiquitinated peptides of STAT3 (with linear Ub sites identified at Lys153, Lys161, Lys163 and Lys191), and the MS/MS spectrum of the characteristic linear Ub peptide (GGMQIFVK) generated by trypsin digestion (shown at the bottom). ( C ) IP analysis of linear ubiquitination of STAT3 in HEK293T cells transfected, respectively, with wild-type STAT3, STAT3-K153R, STAT3-K161R, STAT3-K163R, STAT3-K199R or STAT3-4KR (STAT3-K153/161/163/199R) mutants, and co-transfected with LUBAC. ( D ) STAT3(–/–) HeLa cells were transduced with wild-type STAT3 or the STAT3-4KR mutant, followed by treatment with 10 ng/ml IL-6 for 1 h. Immunoblot analysis of pY705-STAT3 and total STAT3 was performed. ( E ) qPCR analysis of the STAT3-targeted genes SOCS3 and c-Myc in STAT3(–/–) HeLa cells transduced with wild-type STAT3 or the STAT3-4KR mutant after IL-6 treatment (10 ng/ml). Data are presented as the mean ± SD, n = 3, * P <0.05, Student's t -test.

Article Snippet: Human HEK293T cells purchased from the ATCC, and stat3-null HeLa cells purchased from ABclonal (20170728-02) were grown in Dulbecco's modified Eagle’s medium (DMEM) with 10% fetal bovine serum (FBS; FND500, excellbio), 100 U/ml penicillin and 100 U/ml streptomycin.

Techniques: Ubiquitin Proteomics, Residue, Tandem Mass Spectroscopy, Generated, Transfection, Transduction, Mutagenesis, Western Blot

Journal: Materials Today Bio

Article Title: Functionalized DNA-spider silk nanohydrogels for controlled protein binding and release

doi: 10.1016/j.mtbio.2020.100045

Figure Lengend Snippet: Functional analysis of apt-rssp conjugates. (a) Far UV-CD spectra of the aptamer oligonucleotide, rssp and the corresponding conjugate in ion-depleted TBE-buffer (89 ​mM Tris, 89 ​mM boric acid, 20 ​mM EDTA, pH 8.0) as well as Na + /K + rich Gq-buffer (25 ​mM HEPES, pH 8.0, 200 ​mM NaCl, 20 ​mM KCl); (b) SEC analysis of thrombin binding to the conjugates (Yarra SEC-4000 column in Gq-buffer and detection of thrombin-specific tryptophan fluorescence); (c) and (d) TEM of rssp and apt15-rssp fibrils after treatment with thrombin; (e) Fibril self-assembly kinetics of unmodified rssp and corresponding conjugates monitored using turbidity at 340 ​nm as published previously ; (f) Coagulation of fibrin upon fibrinogen-cleavage catalyzed by thrombin without or in the presence of apt-conjugates as indicated. Scale bars 100 ​nm in (c) and (d) and 50 ​nm for the inserts. SEC, size-exclusion chromatography; TEM, transmission electron microscopy; rssp, recombinant spider silk protein.

Article Snippet: Thrombin (5.9 nM) was treated with apt-rssp (400 nM) in the presence of human fibrinogen (Thermo Fisher) (2 μM) and HSA (10 μM) in clotting buffer (25 mM Tris/HCl, pH 7.4, 150 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 1 mM CaCl 2 ).

Techniques: Functional Assay, Circular Dichroism, Binding Assay, Fluorescence, Coagulation, Size-exclusion Chromatography, Transmission Assay, Electron Microscopy, Recombinant

Journal: Antiviral Research

Article Title: Picomolar inhibition of SARS-CoV-2 variants of concern by an engineered ACE2-IgG4-Fc fusion protein

doi: 10.1016/j.antiviral.2021.105197

Figure Lengend Snippet: Structural elements in the ACE2-Fc constructs. a Schematic depiction of the main parts in an engineered ACE2-Fc molecule and their functional properties. b Design of the ACE2-Fc fusion protein; ACE2 parts in light and dark blue, IgG-Fc part in gray, spike (S) protein trimer in green and the receptor-binding domain (RBD) located at the tip of each spike protein in orange and dark green. The binding region as well as active site residues H374 and H378 important for the enzymatic activity of ACE2 are highlighted. Structures of the following Protein Data Bank (PBD) identifiers were used for modeling: 6M17, 6M0J, 6VSB, 5DK3. c Nomenclature and structural variations in the ACE2-Fc constructs. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: 24 h before infection, human lung epithelial A549 cells (ATCC-CCL-185), engineered to overexpress the ACE2 (A549-hACE2), were plated at 1.5E04 cells/well in a 96-well white well half area plate with clear bottom (Corning, Corning, NY, USA) in DMEM containing 2% FCS, 1% P/S and 1% NEAA (all from Gibco) and incubated overnight at 37 °C and 5% CO2.

Techniques: Construct, Functional Assay, Binding Assay, Activity Assay

Journal: Antiviral Research

Article Title: Picomolar inhibition of SARS-CoV-2 variants of concern by an engineered ACE2-IgG4-Fc fusion protein

doi: 10.1016/j.antiviral.2021.105197

Figure Lengend Snippet: Structural and functional characteristics of the ACE2-Fc proteins. a Far-UV CD spectra (left) and Near-UV CD spectra (right) of ACE2-Fc constructs indicating that all proteins exhibit similar secondary and tertiary structures. b Chromatograms and molecular mass from size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS) indicating that the ACE2-Fc molecules form homodimers. c Non-reducing (top) and reducing (bottom) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis showing that intermolecular disulfide bonds in the homodimers are formed. d Comparison of fluorescence signals over time obtained in an assay testing the cleavage of a fluorescent peptidyl-4-methylcoumaryl-7-amide (MCA). Relative fluorescent units (RFU) are given. e Amount of MCA cleaved after 30 min of incubation with the ACE2-Fc constructs. Ref1 and Ref2 are two different commercially available ACE2-Fc proteins from Genscript and Acrobiosystems, respectively. Bars are mean values; error bars depict the 95% confidence interval of six independent experiments shown as circles.

Article Snippet: 24 h before infection, human lung epithelial A549 cells (ATCC-CCL-185), engineered to overexpress the ACE2 (A549-hACE2), were plated at 1.5E04 cells/well in a 96-well white well half area plate with clear bottom (Corning, Corning, NY, USA) in DMEM containing 2% FCS, 1% P/S and 1% NEAA (all from Gibco) and incubated overnight at 37 °C and 5% CO2.

Techniques: Functional Assay, Circular Dichroism, Construct, Size-exclusion Chromatography, Multi-Angle Light Scattering, Polyacrylamide Gel Electrophoresis, SDS Page, Comparison, Fluorescence, Incubation

Journal: Antiviral Research

Article Title: Picomolar inhibition of SARS-CoV-2 variants of concern by an engineered ACE2-IgG4-Fc fusion protein

doi: 10.1016/j.antiviral.2021.105197

Figure Lengend Snippet: Interaction of the ACE2-Fc constructs with the SARS-CoV-2 RBD. a Surface plasmon resonance (SPR) was performed to obtain binding curves of ACE2-Fc fusion constructs and an unrelated Fc fusion protein (aflibercept) to an immobilized RBD from SARS-CoV-2. An exemplary binding curve is shown (RU = Response Units). b Binding constants of the ACE2-Fc constructs towards the RBD of SARS-CoV-2 (Mean ± SD of triplicate measurements). c ACE2-Fc fusion proteins were pre-incubated with the SARS-CoV-2 spike S1 protein and tested in a competition ELISA for their ability to neutralize S1 binding to immobilized ACE2 protein. Potent inhibition of SARS-CoV-2 spike S1 protein by ACE2-IgG4-Fc constructs (left) and ACE2-IgG1-Fc constructs (right). Data are represented as means ± SD of at least two independent experiments.

Article Snippet: 24 h before infection, human lung epithelial A549 cells (ATCC-CCL-185), engineered to overexpress the ACE2 (A549-hACE2), were plated at 1.5E04 cells/well in a 96-well white well half area plate with clear bottom (Corning, Corning, NY, USA) in DMEM containing 2% FCS, 1% P/S and 1% NEAA (all from Gibco) and incubated overnight at 37 °C and 5% CO2.

Techniques: Construct, SPR Assay, Binding Assay, Incubation, Enzyme-linked Immunosorbent Assay, Inhibition

Journal: Antiviral Research

Article Title: Picomolar inhibition of SARS-CoV-2 variants of concern by an engineered ACE2-IgG4-Fc fusion protein

doi: 10.1016/j.antiviral.2021.105197

Figure Lengend Snippet: Binding affinities of ACE2-Fc constructs to Fc-receptors.

Article Snippet: 24 h before infection, human lung epithelial A549 cells (ATCC-CCL-185), engineered to overexpress the ACE2 (A549-hACE2), were plated at 1.5E04 cells/well in a 96-well white well half area plate with clear bottom (Corning, Corning, NY, USA) in DMEM containing 2% FCS, 1% P/S and 1% NEAA (all from Gibco) and incubated overnight at 37 °C and 5% CO2.

Techniques: Binding Assay, Construct

Journal: Antiviral Research

Article Title: Picomolar inhibition of SARS-CoV-2 variants of concern by an engineered ACE2-IgG4-Fc fusion protein

doi: 10.1016/j.antiviral.2021.105197

Figure Lengend Snippet: Effect of ACE2-Fc on SARS-CoV-2-GFP infection and inhibition of SARS-CoV-2 primary isolates. a ACE2-IgG4-Fc reduces SARS-CoV-2-GFP replication. Representative fluorescent images of Vero E6 cells infected with SARS-CoV-2-GFP (multiplicity of infection (MOI) = 0.6 IU/cell) pre-incubated with ACE2-IgG4-Fc fusion construct 1 (632 nM). b ACE2-Fc fusion proteins potently neutralize coronaviruses. Serial dilutions of ACE2-Fc fusion proteins were pre-incubated with different coronaviruses and tested for their ability to neutralize the virus before infection of Vero E6 cells. Neutralization of SARS-CoV (top), SARS-CoV-2-Jan (middle) and SARS-CoV-2-April (bottom) by ACE2-IgG4-Fc constructs (left) and ACE2-IgG1-Fc constructs (right) is shown. Data given are means ± SEM of three independent experiments each. 50% inhibitory concentrations (IC50) determined as well as the 95% confidence interval (CI 95%) are given for each construct. The dashed lines indicate the IC50 values on the corresponding curves.

Article Snippet: 24 h before infection, human lung epithelial A549 cells (ATCC-CCL-185), engineered to overexpress the ACE2 (A549-hACE2), were plated at 1.5E04 cells/well in a 96-well white well half area plate with clear bottom (Corning, Corning, NY, USA) in DMEM containing 2% FCS, 1% P/S and 1% NEAA (all from Gibco) and incubated overnight at 37 °C and 5% CO2.

Techniques: Infection, Inhibition, Incubation, Construct, Virus, Neutralization

Journal: Antiviral Research

Article Title: Picomolar inhibition of SARS-CoV-2 variants of concern by an engineered ACE2-IgG4-Fc fusion protein

doi: 10.1016/j.antiviral.2021.105197

Figure Lengend Snippet: Neutralization potency of ACE2-IgG4-Fc fusion proteins increases with evolution of pandemic SARS-CoV-2 variants. Serial dilutions of ACE2-IgG4-Fc fusion constructs 1 and 3 were pre-incubated with the indicated SARS-CoV-2 primary isolates or VoCs and tested for their ability to prevent cytotoxicity following infection of A549-hACE2 cells. Neutralization of SARS-CoV-2-Jan, SARS-CoV-2-April and SARS-CoV-2 VoCs alpha, beta and delta by enzymatically active ACE2-IgG4-Fc construct 1 (left) and enzymatically inactive ACE2-IgG4-Fc construct 3 (right). Data given are means ± SEM of three independent experiments each. 50% inhibitory concentrations (IC50 values) determined as well as the 95% confidence interval (CI 95%) are shown for each construct. The dashed lines indicate the IC50 values on the corresponding curves.

Article Snippet: 24 h before infection, human lung epithelial A549 cells (ATCC-CCL-185), engineered to overexpress the ACE2 (A549-hACE2), were plated at 1.5E04 cells/well in a 96-well white well half area plate with clear bottom (Corning, Corning, NY, USA) in DMEM containing 2% FCS, 1% P/S and 1% NEAA (all from Gibco) and incubated overnight at 37 °C and 5% CO2.

Techniques: Neutralization, Construct, Incubation, Infection

Journal: Molecular & Cellular Proteomics

Article Title: Nucleolin Mediates MicroRNA-directed CSF-1 mRNA Deadenylation but Increases Translation of CSF-1 mRNA

doi: 10.1074/mcp.m112.025288

Figure Lengend Snippet: FIG. 2. Nucleolin binds to the G-quadruplex and ARE in CSF-1 mRNA 3UTR in vitro. A and B, IP of nucleolin-RNA complexes from Hey cell lysates. IP assays were carried out using anti-human nucleolin mAb or IgG. The presence of nucleolin in the IP materials was monitored by IB. qRT-PCR measurements of CSF-1 mRNA in nucleolin IP show direct interaction between nucleolin and CSF-1 mRNA. Bcl-xL mRNA in nucleolin IP serves as a positive control. The mean S.D. of CSF-1 mRNA normalized for actin mRNA is depicted (n 3). Actin mRNA was set to equal 1. C, in vitro binding assay by UV cross-linking shows recombinant nucleolin binds to the G-quadruplex (WT) and mutant sequences (Mut) in the CSF-1 RNA 3UTR 50-nt sequence in the presence of 100 mM KCl. In the absence of KCl, no binding of wild type (Wt) sequence to recombinant nucleolin was detected. Mutation in region 3 GGG to UUU (Mut-3) abrogated nucleolin binding. CD spectra showing the G-quadruplex formation in CSF-1 RNA 3UTR (WT, Mut-1, and Mut-5) in the presence of 100 mM KCl. CD spectra of Mut-2, -3, -4, and -6 show formation of unknown structures. D, EMSA shows recombinant nucleolin associates with the CSF-1 3UTR G-quadruplex RNA. The 32P-labeled CSF-1 3UTR G-quadruplex riboprobe (48 nt, 2,850–2,897 nt) from 3UTR associated with nucleolin and shifted in native PAGE. MBP, used as a negative control, was not associated with CSF-1 3UTR G-quadruplex RNA. E, in vitro binding assay by UV cross-linking shows nucleolin binds to the ARE in the CSF-1 RNA 3UTR 144-nt sequence. No binding of CSF-1 mRNA coding region (CR) to recombinant nucleolin was detected. MBP, used as a negative control, was not bound to CSF-1 RNA.

Article Snippet: For nucleolin IP, 5 g of mouse monoclonal antihuman nucleolin antibody (sc-17826, Santa Cruz Biotechnology) was used.

Techniques: In Vitro, Quantitative RT-PCR, Positive Control, Binding Assay, Recombinant, Mutagenesis, Sequencing, Circular Dichroism, Labeling, Clear Native PAGE, Negative Control

Journal: Molecular & Cellular Proteomics

Article Title: Nucleolin Mediates MicroRNA-directed CSF-1 mRNA Deadenylation but Increases Translation of CSF-1 mRNA

doi: 10.1074/mcp.m112.025288

Figure Lengend Snippet: FIG. 3. Nucleolin enhances deadeny- lation of CSF-1 mRNA but increases CSF-1 mRNA translation. A, nucleolin overexpression increases CSF-1 protein levels in Bix3 and SKOV3 ovarian cancer cells. B and C, when the cDNA was syn- thesized by oligo(dT)18, nucleolin over- expression decreases CSF-1 mRNA lev- els in Bix3 and SKOV3 ovarian cancer cells. D, CSF-1 mRNA stability is de- creased by nucleolin overexpression when cDNA was synthesized by oli- go(dT)18 in SKOV3 cells. E, when the cDNA was synthesized by random primer (pdN6), nucleolin overexpression increases CSF-1 mRNA levels in Bix3 cells. F and G, in SKOV3 cells, when the cDNA was synthesized by random primer (pdN6), there was no significant effect of nucleolin overexpression on CSF-1 mRNA or mRNA stability. This suggests an effect of nucleolin on CSF-1 translation in these cells. B–G, n 3. Mean S.D. is presented. Deadenyla- tion assay of luciferase RNA fused with CSF-1 mRNA 3UTR sequences (H) or endogenous CSF-1 mRNA, by ligation- mediated poly(A) test (I) is shown. J, nucleolin abundance affects the distri- bution of CSF-1 mRNA on polysomes. SKOV3 cells were transfected with either nucleolin-EGFP or EGFP. Polysome pro- files were prepared with the sucrose density gradient ultracentrifugation. Rel- ative distributions of CSF-1 mRNA. K, GAPDH mRNA in polysome gradients after nucleolin overexpression. L, relative distributions of nucleolin and -tubulin were obtained by IB analyses of the polysome gradient. Fractions 1 and 2, unbound RNPs (Unb.); fractions 3 and 4, monosomes (Mono.); and fractions 5–10, low and high molecular weight polysomes (LMW and HMW).

Article Snippet: For nucleolin IP, 5 g of mouse monoclonal antihuman nucleolin antibody (sc-17826, Santa Cruz Biotechnology) was used.

Techniques: Over Expression, Synthesized, Luciferase, Ligation, Transfection, High Molecular Weight

Journal: Molecular & Cellular Proteomics

Article Title: Nucleolin Mediates MicroRNA-directed CSF-1 mRNA Deadenylation but Increases Translation of CSF-1 mRNA

doi: 10.1074/mcp.m112.025288

Figure Lengend Snippet: FIG. 5. miRNA-directed CSF-1 mRNA decay is dependent on nucleolin. A and D, overexpression of miR-130a and miR-301a reduced CSF-1 mRNA levels in Bix3 (p 0.026 and 0.024, respectively) and in SKOV3 cells (p 0.021 and 0.01, respectively). B and E, inhibition of miR-130a and miR-301a increased CSF-1 mRNA levels in Bix3 and SKOV3 cells. C and F, immunoblot analysis of CSF-1 protein in either miR-130a or miR-301a overexpressed or inhibited Bix3 and SKOV3 cells. G and H, silencing of nucleolin in miR-130a or miR-301a overexpressed cells did not decrease CSF-1 mRNA and protein levels in SKOV3 cells. I and J, at 2 days after transfection, Bix3 and SKOV3 cells were treated with Act-D, and the half-life of CSF-1 mRNA was measured by qRT-PCR. I, half-life of CSF-1 mRNA was 4.3 h. Overexpression of miR-130a reduced the half-life to 2.4 h. Silencing of nucleolin in miR-130a-overexpressed cells recovered the half-life to 3.4 h. J, overexpression of miR-301a reduced the half-life of CSF-1 mRNA to 2.6 h. Silencing of nucleolin in miR-301a overexpressed cells recovered the half-life to 3.4 h. n 3. Mean S.D. is presented.

Article Snippet: For nucleolin IP, 5 g of mouse monoclonal antihuman nucleolin antibody (sc-17826, Santa Cruz Biotechnology) was used.

Techniques: Over Expression, Inhibition, Western Blot, Transfection, Quantitative RT-PCR

Journal: Molecular & Cellular Proteomics

Article Title: Nucleolin Mediates MicroRNA-directed CSF-1 mRNA Deadenylation but Increases Translation of CSF-1 mRNA

doi: 10.1074/mcp.m112.025288

Figure Lengend Snippet: FIG. 6. miR-130a and miR-301a in- hibit in vitro motility of ovarian cancer cells, an effect that is dependent on nucleolin. After transfection with the in- dicated construct(s), Hey cells were plated on an 8-m pore membrane for a 6-h directed motility assay. Mean S.E. is depicted. n 4.

Article Snippet: For nucleolin IP, 5 g of mouse monoclonal antihuman nucleolin antibody (sc-17826, Santa Cruz Biotechnology) was used.

Techniques: In Vitro, Transfection, Construct, Membrane, Motility Assay

Journal: Molecular & Cellular Proteomics

Article Title: Nucleolin Mediates MicroRNA-directed CSF-1 mRNA Deadenylation but Increases Translation of CSF-1 mRNA

doi: 10.1074/mcp.m112.025288

Figure Lengend Snippet: FIG. 7. Nucleolin domains differen- tially regulate CSF-1 mRNA and pro- tein expression. A, schematic diagram of nucleolin functional domains. Expres- sion constructs containing wild type nucleolin or each domain-tagged by EGFP were generated. Wild type nucleo- lin or the individual functional domain was transiently transfected in SKOV3 (B, D, F, and H) and Bix3 (C, E, G, and I) ovarian cancer cells. In both cells (B and C), introduction of the RRM domain in- creased CSF-1 mRNA levels dramati- cally. In contrast, the presence of acidic or RGG domains did not influence CSF-1 mRNA levels significantly. n 3. Mean S.D. is presented. D and E, in- troduction of acidic and RGG domains decreased, whereas the RRM domain in- creased CSF-1 protein levels. F and G, immunoblot of transiently transfected SKOV3 and Bix3 ovarian cancer cells by nucleolin Ab. Endogenous nucleolin is shown at 110 kDa. Only endogenous nucleolin and RRM domain can be de- tected by the nucleolin Ab, which is raised by the RRM domain epitope. H and I, immunoblot of transiently trans- fected SKOV3 and Bix3 ovarian cancer cells by EGFP Ab.

Article Snippet: For nucleolin IP, 5 g of mouse monoclonal antihuman nucleolin antibody (sc-17826, Santa Cruz Biotechnology) was used.

Techniques: Expressing, Functional Assay, Construct, Generated, Transfection, Western Blot

Journal: Molecular & Cellular Proteomics

Article Title: Nucleolin Mediates MicroRNA-directed CSF-1 mRNA Deadenylation but Increases Translation of CSF-1 mRNA

doi: 10.1074/mcp.m112.025288

Figure Lengend Snippet: FIG. 8. Interaction of nucleolin with Ago2 and PABPC. A, co-IP assay from Hey cell lysates. Co-IP assay was carried out using anti-human nucleolin mAb or IgG. The presence of nucleolin and Ago2 in the IP materials was monitored by IB. Nucleolin and Ago2 are coimmunopre- cipitated in the presence of RNase inhibitor. In contrast, in the presence of RNase A and T1, nucleolin and Ago2 are not coimmunoprecipitated, indicating an indirect interaction between nucleolin and Ago2 via mRNA. B, co-IP assay from Hey cell lysates. Co-IP assay was carried out using anti-human PABPC mAb or IgG. Nucleolin and PABPC are coimmunoprecipitated in either presence of RNase inhibitor or RNase A and T1, indicating a direct interaction between PABPC and nucleolin. C, size-exclusion chromatography of SKOV3 cell lysates. Nucleolin, Ago2, and PABPC are eluted together in fractions 10–16, in which CSF-1 mRNA is present.

Article Snippet: For nucleolin IP, 5 g of mouse monoclonal antihuman nucleolin antibody (sc-17826, Santa Cruz Biotechnology) was used.

Techniques: Co-Immunoprecipitation Assay, Size-exclusion Chromatography

Journal: Molecular & Cellular Proteomics

Article Title: Nucleolin Mediates MicroRNA-directed CSF-1 mRNA Deadenylation but Increases Translation of CSF-1 mRNA

doi: 10.1074/mcp.m112.025288

Figure Lengend Snippet: FIG. 9. Model for post-transcriptional regulation of CSF-1 mRNA and protein expression. A, formation of nucleolin-Ago2-PABPC complex induces deadenylation of CSF-1 mRNA. B, nucleolin in association with PABPC activates translation by formation of the cap-de- pendent mRNP closed loop.

Article Snippet: For nucleolin IP, 5 g of mouse monoclonal antihuman nucleolin antibody (sc-17826, Santa Cruz Biotechnology) was used.

Techniques: Expressing

Journal: Frontiers in Endocrinology

Article Title: Goldfish adiponectin: (I) molecular cloning, tissue distribution, recombinant protein expression, and novel function as a satiety factor in fish model

doi: 10.3389/fendo.2023.1283298

Figure Lengend Snippet: Domain organization and 3D protein modeling of goldfish AdipoQ. (A) Domain organization of the goldfish AdipoQ precursor compared with its counterparts in other vertebrates. The percentage similarity (%Similarity) for protein sequences of individual domains in the AdipoQ precursor of other species compared with the corresponding structures in goldfish AdipoQ were deduced by Clustal W with scoring > 0.5 using the Gonnet PAM250 matrix. (B) Comparison of 3D protein structure of goldfish AdipoQ precursor with its human counterpart. The ribbon plots for the 3D models of goldfish and human AdipoQ precursors were deduced by SWISS-MODEL with the anti-parallel β sheets labeled in red, α helical segments labeled in green, and random coil structures labeled in pink. The corresponding surface plots for charge distribution were constructed by Chimera 1.16 with the acidic residues carrying negative charge labeled in red, basic residues carrying positive charge labeled in blue, and hydrophobic residues with little/no charge labeled in white/light grey.

Article Snippet: For central expression of the AdipoQ receptor at the protein level, Western blot was also conducted in goldfish brain lysate using the antisera for mouse AdipoR1 (1:1,000) and AdipoR2 (1:4,000), respectively (Aviva Systems Biology, San Diego, CA).

Techniques: Comparison, Labeling, Construct

Journal: Frontiers in Endocrinology

Article Title: Goldfish adiponectin: (I) molecular cloning, tissue distribution, recombinant protein expression, and novel function as a satiety factor in fish model

doi: 10.3389/fendo.2023.1283298

Figure Lengend Snippet: Tissue distribution, phylogenetic analysis, and intron/exon organization of goldfish AdipoQ. (A) Expression profiling of AdipoQ in different tissues and selected brain areas in goldfish using RT-PCR. The authenticity of PCR products detected was confirmed by PCR Southern using a DIG-labeled cDNA probe for goldfish AdipoQ and parallel PCR for β actin was used as an internal control. (B) Phylogenetic analysis of goldfish AdipoQ with the corresponding sequences found in other vertebrates using MEGA X with minimum evolution method. The guide tree was constructed with PHYLIP 2.0 with the percentage of bootstrap values (based on 1,000 bootstraps) presented in individual nodes, and the scale bar represents the phylogenetic distance of evolution. (C) Comparison of intron/exon organization in goldfish AdipoQ gene with the corresponding sequences in other vertebrates. The gene sequences for AdipoQ in representative species from fish to mammals were downloaded from the NCBI genome database and analyzed with Splicing Finder 2.4.1 to deduce the intron/exon junctions for structural comparison of AdipoQ genes in different species.

Article Snippet: For central expression of the AdipoQ receptor at the protein level, Western blot was also conducted in goldfish brain lysate using the antisera for mouse AdipoR1 (1:1,000) and AdipoR2 (1:4,000), respectively (Aviva Systems Biology, San Diego, CA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Labeling, Control, Construct, Comparison

Journal: Frontiers in Endocrinology

Article Title: Goldfish adiponectin: (I) molecular cloning, tissue distribution, recombinant protein expression, and novel function as a satiety factor in fish model

doi: 10.3389/fendo.2023.1283298

Figure Lengend Snippet: Comparative synteny of AdipoQ genes in the respective chromosomes from fish to mammals. The genomic scaffolds with AdipoQ gene in the respective chromosomes of different species were downloaded from the NCBI genome database. The neighboring genes located upstream/downstream of AdipoQ gene were curated using Genomicus software. Based on the similarity of syntenic genes around the AdipoQ gene in different species, three clusters of evolution lineages related to AdipoQ gene can be discerned, including the fish lineage (goldfish and zebrafish), amphibian/reptilian/avian lineage (Xenopus, lizard, and chicken), and mammalian lineage (mouse and human). The syntenic genes located in the neighborhood of AdipoQ gene in the same genomic scaffold are presented in the form of colored polygons with the pointed end indicating the transcriptional orientation. The ortholog genes in the same vertebrate class/evolution lineage are linked with black lines whereas the AdipoQ and ST6GAL1 genes, which can be found in different vertebrate classes/evolution lineages, are linked with green lines.

Article Snippet: For central expression of the AdipoQ receptor at the protein level, Western blot was also conducted in goldfish brain lysate using the antisera for mouse AdipoR1 (1:1,000) and AdipoR2 (1:4,000), respectively (Aviva Systems Biology, San Diego, CA).

Techniques: Software

Journal: Frontiers in Endocrinology

Article Title: Goldfish adiponectin: (I) molecular cloning, tissue distribution, recombinant protein expression, and novel function as a satiety factor in fish model

doi: 10.3389/fendo.2023.1283298

Figure Lengend Snippet: Recombinant goldfish AdipoQ expression, purification, characterization, and functional testing. (A) Expression of His-tagged goldfish AdipoQ in E coli followed by IMAC purification. Samples obtained at different stages of purification were resolved by SDS-PAGE followed by Coomassie blue staining. The identity of His-tagged AdipoQ expressed was confirmed by Western blot using an anti-His antibody (Anti-His Ab) (UI, uninduced bacteria culture; ID, bacteria culture induced with 1 mM IPTG; P, pellet fraction of induced culture; S, soluble fraction of induced culture; F, flow-through of soluble fraction after passing through IMAC column; W, Washout fraction with 60 mM imidazole; E, eluate with product released by 300 mM imidazole). (B) Size exclusion chromatography (SEC) for further purification of goldfish AdipoQ. Recombinant AdipoQ eluted from the IMAC column was loaded into a HiLoad 26/60 S200 SEC column, and the protein fractions covering the protein peak were then used for SDS-PAGE followed by Coomassie blue staining. (C) Evaluation of apparent MW in solution for recombinant AdipoQ using size exclusion chromatography coupled with multiangle laser light scattering (SEC-MALS). The top five protein fractions obtained from the preceding study were pooled, and a 200-μl sample of the final product was subjected to a second round of SEC using a S12 10/300 GL column linked with a DynaPro NanoStar Dynamic Light Scattering Detector. In this case, a protein peak with an average MW of 56 kDa (~3 folds of the AdipoQ monomer) could be detected by SEC-MALS analysis. (D) Characterization of recombinant AdipoQ with PAGE in native gel under non-denaturing conditions. Parallel SDS-PAGE under denaturing conditions was used as a reference for the monomeric form of AdipoQ. Under non-denaturing conditions, the trimeric, hexameric, and HMW forms of AdipoQ could be detected with the trimeric AdipoQ as the dominant product. (E) Structural analysis of recombinant goldfish AdipoQ using circular dichroism (CD). Goldfish AdipoQ produced was subjected to far UV CD analysis to evaluate the secondary structures (α helix/β sheet) found in goldfish AdipoQ (with subtraction of the background signal of the solvent used). (F) Protein modeling of the trimeric complex formed by the globular domains of goldfish AdipoQ compared with the corresponding structure in human. 3D protein modeling of goldfish gAdipoQ was constructed using SWISS-MODEL with the crystal structure of human gAdipoQ as a reference. (G) Function testing of recombinant AdipoQ in HepG2 cells with AdipoQ receptor (AdipoR) expression. After co-transfection of different components of the PPRE reporter system (for probing AMPK activation) with the expression vector for goldfish AdipoR (AdipoR1a, AdipoR1b, and AdipoR2, respectively), HepG2 cells were challenged for 18 h with increasing doses (1–100 nM) of recombinant AdipoQ and with parallel treatment of metformin (1 mM) as the positive control. After that, luciferase activities expressed were measured using a dual luciferase assay kit. Groups denoted with different letters represent a significant difference at p < 0.05 (one-way ANOVA followed by Newman–Keuls test).

Article Snippet: For central expression of the AdipoQ receptor at the protein level, Western blot was also conducted in goldfish brain lysate using the antisera for mouse AdipoR1 (1:1,000) and AdipoR2 (1:4,000), respectively (Aviva Systems Biology, San Diego, CA).

Techniques: Recombinant, Expressing, Purification, Functional Assay, SDS Page, Staining, Western Blot, Bacteria, Size-exclusion Chromatography, Circular Dichroism, Produced, Solvent, Construct, Cotransfection, Activation Assay, Plasmid Preparation, Positive Control, Luciferase

Journal: Frontiers in Endocrinology

Article Title: Goldfish adiponectin: (I) molecular cloning, tissue distribution, recombinant protein expression, and novel function as a satiety factor in fish model

doi: 10.3389/fendo.2023.1283298

Figure Lengend Snippet: Association of AdipoQ with food intake and feeding regulation in goldfish. Effects of food intake in goldfish on plasma AdipoQ (A) and AdipoQ gene expression in the liver (B) , telencephalon (C) , optic tectum (D) , hypothalamus (E) , and muscle (F) , respectively. Goldfish entrained with a “one-meal-per-day” feeding schedule was divided into two groups, with one group as “Fed” group with food pellets provided at 10:00 AM (as time zero) and the other group as “Unfed” group without food provision (as the control). Plasma samples and selected tissues were harvested at the time points as indicated. Plasma AdipoQ was measured by FIA, and AdipoQ transcript level was monitored by real-time PCR. Time course (G) and dose dependence (H) of AdipoQ treatment on surface foraging, bottom foraging, and food spitting in goldfish. IP injection of recombinant goldfish AdipoQ (300 ng/g BW for time course and 100–500 ng/g BW for dose dependence) was conducted 10 min prior to the scheduled feeding time (at 10:00 AM), and different types of feeding behaviors were scored over a 2-h period after the introduction of food pellets. Parallel injection of physiological saline was used as the control. In the study for dose dependence of AdipoQ action, food consumption was also monitored by the end of the 2-h period. Groups denoted by asterisks (Student’s t test compared with time-matched control) or with different letters (two-way ANOVA followed by Bonferroni test for time course and one-way ANOVA followed by Newman–Keuls test for dose dependence study) represent a significant difference at p < 0.05.

Article Snippet: For central expression of the AdipoQ receptor at the protein level, Western blot was also conducted in goldfish brain lysate using the antisera for mouse AdipoR1 (1:1,000) and AdipoR2 (1:4,000), respectively (Aviva Systems Biology, San Diego, CA).

Techniques: Clinical Proteomics, Gene Expression, Control, Real-time Polymerase Chain Reaction, Injection, Recombinant, Saline

Journal: Frontiers in Endocrinology

Article Title: Goldfish adiponectin: (I) molecular cloning, tissue distribution, recombinant protein expression, and novel function as a satiety factor in fish model

doi: 10.3389/fendo.2023.1283298

Figure Lengend Snippet: IP injection of AdipoQ on central expression of orexigenic/anorexigenic factors in brain areas for feeding control in goldfish. Goldfish with prior training of “one-meal-per-day” feeding schedule were IP injected with recombinant AdipoQ (300 ng/g BW) at the scheduled feeding time without food provision (as time zero). Parallel injection with physiological saline was used as the control. After IP injection, brain areas including the telencephalon, hypothalamus, and optic tectum were harvested at the time points as indicated. Total RNA was isolated and used for real-time PCR to monitor the transcript expression for (A) orexigenic factors including NPY, AgRP, Orexin, and Apelin, and (B) anorexigenic factors including CCK, POMC, CART, and MCH. Parallel real-time PCR for 18S RNA was used as the internal control. Groups denoted by different letters represent a significant difference at p < 0.05.

Article Snippet: For central expression of the AdipoQ receptor at the protein level, Western blot was also conducted in goldfish brain lysate using the antisera for mouse AdipoR1 (1:1,000) and AdipoR2 (1:4,000), respectively (Aviva Systems Biology, San Diego, CA).

Techniques: Injection, Expressing, Control, Recombinant, Saline, Isolation, Real-time Polymerase Chain Reaction

Journal: Frontiers in Endocrinology

Article Title: Goldfish adiponectin: (I) molecular cloning, tissue distribution, recombinant protein expression, and novel function as a satiety factor in fish model

doi: 10.3389/fendo.2023.1283298

Figure Lengend Snippet: IP injection of AdipoQ on central expression of the receptors for feeding regulators and hepatic expression of leptin and ghrelin in goldfish. IP injection of AdipoQ (300 ng/g BW) was performed with parallel treatment of physiological saline as the control. After that, the liver as well as brain areas including the telencephalon, hypothalamus, and optic tectum were harvested at the time points as indicated. Total RNA was isolated from brain areas collected and used for real-time PCR to monitor transcript expression of (A) the receptors for orexigenic factors including GHSR 1A1 , GHSR 1A2 , and NPY1R, and (B) the receptors for anorexigenic factors including LepR and MC4R. Similar operation was conducted in the liver samples with real-time PCR for (C) the peripheral feeding regulators including leptin A1, leptin A2, and ghrelin. In this study, real-time PCR for 18S RNA was used as the internal control. Groups denoted by different letters represent a significant difference at p < 0.05.

Article Snippet: For central expression of the AdipoQ receptor at the protein level, Western blot was also conducted in goldfish brain lysate using the antisera for mouse AdipoR1 (1:1,000) and AdipoR2 (1:4,000), respectively (Aviva Systems Biology, San Diego, CA).

Techniques: Injection, Expressing, Saline, Control, Isolation, Real-time Polymerase Chain Reaction

Journal: Frontiers in Endocrinology

Article Title: Goldfish adiponectin: (I) molecular cloning, tissue distribution, recombinant protein expression, and novel function as a satiety factor in fish model

doi: 10.3389/fendo.2023.1283298

Figure Lengend Snippet: Expression of AdipoQ and its receptors within the brain and central actions of AdipoQ on feeding behaviors and food consumption in goldfish. (A) Co-localization of AdipoQ and its receptors AdipoR1a, AdipoR1b, and AdipoR2 in brain areas involved in feeding control as revealed by RT-PCR. In this study, the brain was used as the positive control and parallel PCR without template was used as a negative control. RT-PCR targeting β actin expression was used as the internal control. (B) Detection of AdipoR1 and R2 immunoreactivities in goldfish brain and liver using Western blot. Using the antibodies (Ab) raised against mouse AdipoR1 and R2, respectively, Western blot was conducted in tissue lysates prepared from the brain and liver of the goldfish, respectively. The specificity of AdipoR1/R2 signals detected was also confirmed by parallel Western blot using the same antibodies preabsorbed with the respective antigen peptides used to raise the two antibodies provided by the company. In this study, Western blot for β actin was conducted to serve as a loading control. (C) Time course and (D) dose dependence of central administration with AdipoQ on surface foraging, bottom foraging, and food spitting in goldfish. ICV injection of recombinant AdipoQ (300 ng/g BW for time course and 100–500 ng/g BW for dose dependence) was conducted 15 min prior to the scheduled feeding time, and different types of feeding behaviors were scored over a 2-h period after the introduction of food pellets. Parallel ICV injection of physiological saline was used as the control treatment. In the study for dose dependence of AdipoQ treatment, food consumption was also monitored by the end of the 2-h period. Groups denoted by asterisks or with different letters represent a significant difference at p < 0.05.

Article Snippet: For central expression of the AdipoQ receptor at the protein level, Western blot was also conducted in goldfish brain lysate using the antisera for mouse AdipoR1 (1:1,000) and AdipoR2 (1:4,000), respectively (Aviva Systems Biology, San Diego, CA).

Techniques: Expressing, Control, Reverse Transcription Polymerase Chain Reaction, Positive Control, Negative Control, Western Blot, Injection, Recombinant, Saline

Journal: Frontiers in Endocrinology

Article Title: Goldfish adiponectin: (I) molecular cloning, tissue distribution, recombinant protein expression, and novel function as a satiety factor in fish model

doi: 10.3389/fendo.2023.1283298

Figure Lengend Snippet: ICV injection of AdipoQ on central expression of orexigenic/anorexigenic factors in brain areas for feeding control in goldfish. Goldfish with prior training of “one-meal-per-day” feeding schedule were ICV injected with recombinant AdipoQ (300 ng/g BW) 15 min prior to the scheduled feeding time without food provision (as time zero). Parallel treatment with physiological saline was used as the control. After ICV injection, the brain areas including the telencephalon, hypothalamus, and optic tectum were harvested at the time points as indicated. Total RNA was isolated and subjected to real-time PCR for transcript expression of (A) orexigenic factors including NPY, AgRP, Orexin, and Apelin, and (B) anorexigenic factors including POMC, CCK, CART, and MCH. Real-time PCR for 18S RNA was also conducted to serve as the internal control. Groups denoted by different letters represent a significant difference at p < 0.05.

Article Snippet: For central expression of the AdipoQ receptor at the protein level, Western blot was also conducted in goldfish brain lysate using the antisera for mouse AdipoR1 (1:1,000) and AdipoR2 (1:4,000), respectively (Aviva Systems Biology, San Diego, CA).

Techniques: Injection, Expressing, Control, Recombinant, Saline, Isolation, Real-time Polymerase Chain Reaction

Journal: Frontiers in Endocrinology

Article Title: Goldfish adiponectin: (I) molecular cloning, tissue distribution, recombinant protein expression, and novel function as a satiety factor in fish model

doi: 10.3389/fendo.2023.1283298

Figure Lengend Snippet: ICV injection of AdipoQ on receptor expression for orexigenic/anorexigenic signals in brain areas for feeding control in goldfish. ICV injection of AdipoQ (300 ng/g BW) was performed as described in preceding figure with parallel treatment of physiological saline as the control. After that, brain areas including the telencephalon, hypothalamus, and optic tectum were harvested at the time points as indicated. Total RNA was isolated and subjected to real-time PCR for transcript expression of (A) the receptors for orexigenic signals including GHSR 1A1 , GHSR 1A2 , and NPY1R, and (B) the receptors for anorexigenic signals including LepR and MC4R. Real-time PCR for 18S RNA was used as internal control, and groups denoted by different letters represent a significant difference at p < 0.05.

Article Snippet: For central expression of the AdipoQ receptor at the protein level, Western blot was also conducted in goldfish brain lysate using the antisera for mouse AdipoR1 (1:1,000) and AdipoR2 (1:4,000), respectively (Aviva Systems Biology, San Diego, CA).

Techniques: Injection, Expressing, Control, Saline, Isolation, Real-time Polymerase Chain Reaction

Journal: Frontiers in Endocrinology

Article Title: Goldfish adiponectin: (I) molecular cloning, tissue distribution, recombinant protein expression, and novel function as a satiety factor in fish model

doi: 10.3389/fendo.2023.1283298

Figure Lengend Snippet: Working model for AdipoQ as a novel satiety factor in goldfish. In goldfish, food intake triggers AdipoQ signals both in the liver and within the brain. For the central responses, food intake can stimulate AdipoQ expression in brain areas involved in feeding control including the telencephalon, optic tectum, and hypothalamus. Through AdipoR1/R2 activation, AdipoQ expressed in these brain areas acts locally to (i) upregulate orexigenic signals including NPY, AgRP, orexin, and apelin, (ii) suppress anorexigenic signals including POMC, CCK, CART, and MCH, and (iii) differentially regulate receptor expression of orexigenic/anorexigenic signals, with a rise in the receptors mediating feeding inhibition (LepR and MC4R) but a drop in the receptors for feeding stimulation (NPY1R and GHSR 1A1 & 1A2 ). In addition to the central actions, food intake can also induce AdipoQ expression in the liver with a subsequent rise of AdipoQ secretion into systemic circulation. The endocrine signal of AdipoQ probably can pass through the blood–brain barrier (BBB) and act together with the central signals of AdipoQ to suppress different types of foraging behaviors observed in goldfish. Meanwhile, AdipoQ produced at the hepatic level may act locally to stimulate leptin A1 and A2 expression with a concurrent drop in ghrelin expression in the liver. The corresponding changes in leptin and ghrelin output from the liver via subsequent endocrine actions in the brain may further enhance/prolong the feeding inhibition caused by AdipoQ signals.

Article Snippet: For central expression of the AdipoQ receptor at the protein level, Western blot was also conducted in goldfish brain lysate using the antisera for mouse AdipoR1 (1:1,000) and AdipoR2 (1:4,000), respectively (Aviva Systems Biology, San Diego, CA).

Techniques: Expressing, Control, Activation Assay, Inhibition, Produced

Journal: Frontiers in Endocrinology

Article Title: Goldfish adiponectin: (I) molecular cloning, tissue distribution, recombinant protein expression, and novel function as a satiety factor in fish model

doi: 10.3389/fendo.2023.1283298

Figure Lengend Snippet:

Article Snippet: For central expression of the AdipoQ receptor at the protein level, Western blot was also conducted in goldfish brain lysate using the antisera for mouse AdipoR1 (1:1,000) and AdipoR2 (1:4,000), respectively (Aviva Systems Biology, San Diego, CA).

Techniques: Circular Dichroism, Affinity Chromatography, Size-exclusion Chromatography, Injection, Fluorescence, Reverse Transcription