Journal: EMBO Reports

Article Title: CTRP9 engages AdipoR1 and promotes T cell glycolysis and immunity

doi: 10.1038/s44319-025-00640-0

Figure Lengend Snippet: ( A ) Prediction of the tertiary structure of AdipoR1 from mouse and tilapia. ( B ) A phylogenetic tree for AdipoR1 was constructed with the amino acid sequences of AdipoR1 from the indicated species. The tree was constructed in MEGA7 by using the neighbor-joining (NJ) method with 1000 bootstrap replications. ( C ) Copy numbers of ADPN and its receptor genes in tilapias and other fish species. ( D , E ) Collinearity analysis of ADPNa ( D ) and ADPNb ( E ) based on genomics of the Nile tilapia and other fish species. ( F ) Prediction of the tertiary structure of CTRP9 from mouse and tilapia. ( G ) Global docking results for the CTRP9–AdipoR1 complex obtained using the ZDOCK server. Green sticks indicate salt bridges, and blue and pink sticks indicate CTRP-9 and AdipoR1, respectively. ( H , I ) Identification of tilapia AdipoR1 antibodies. Western blot using tilapia leukocytes showed the specificity of AdipoR1 antibody ( H ). Flow cytometry showing AdipoR1 staining in gated lymphocytes of tilapia ( I ). ( J ) Spleen leukocytes were incubated with recombinant His-tag CTRP9 and then stained with anti-His and anti-AdipoR1 antibody. Flow cytometry showing the frequency of CTRP9-binding cells among AdipoR1 + or AdipoR1 - lymphocytes. Data information: Experiments in ( H – J ) were repeated three times.

Article Snippet: For in vitro activation of AdipoR1, the spleen leukocytes cultured in DMEM (10% FBS, 1% penicillin/streptomycin) were treated with 10 μM AdipoRon (MedChemExpress) for the indicated time.

Techniques: Construct, Western Blot, Flow Cytometry, Staining, Incubation, Recombinant, Binding Assay

Journal: EMBO Reports

Article Title: CTRP9 engages AdipoR1 and promotes T cell glycolysis and immunity

doi: 10.1038/s44319-025-00640-0

Figure Lengend Snippet: Tilapia individuals were infected with A. hydrophila , and spleen leukocytes were isolated at the indicated times. ( A ) Relative mRNA levels of AdipoR1 by qPCR on 3 and 5 dpi, n = 4. ( B ) Protein levels of AdipoR1 by Western blot on 4 and 7 dpi. ( C , D ) Representative FACS plots ( C ) and bar figure ( D ) showing the AdipoR1 + cells in gated lymphocytes, n = 4. ( E ) Immunofluorescence analysis showing the AdipoR1 + cells at the indicated dpi. ( F , G ) Representative FACS plots ( F ) and bar figure ( G ) showing the CD3 and AdipoR1 staining in gated lymphocytes from uninfected individuals, n = 4. ( H – K ) Representative FACS plots ( H , J ) and bar figures ( I , K ) showing the CD3 staining in gated AdipoR1 + or AdipoR1 - cells on 5 dpi, n = 4. ( L ) Representative FACS plots showing the CD3 staining in gated lymphocytes on 5 dpi. ( M , N ) Representative FACS plots ( M ) and bar figures ( N ) showing AdipoR1 staining in gated CD3 + or CD3 - cells on 5 dpi, n = 4. Data information: n stands for biological replicates. Error bars indicate mean ± SEM. Significance between the groups was determined by a two-tailed Student’s t test.

Article Snippet: For in vitro activation of AdipoR1, the spleen leukocytes cultured in DMEM (10% FBS, 1% penicillin/streptomycin) were treated with 10 μM AdipoRon (MedChemExpress) for the indicated time.

Techniques: Infection, Isolation, Western Blot, Immunofluorescence, Staining, Two Tailed Test

Journal: EMBO Reports

Article Title: CTRP9 engages AdipoR1 and promotes T cell glycolysis and immunity

doi: 10.1038/s44319-025-00640-0

Figure Lengend Snippet: Tilapia individuals infected with A. hydrophila were i.p. injected with purified AdipoR1 antibody on days 2, 3, 4, and 5, and the animals were sacrificed at the indicated time points for assay. ( A – K ) Spleen leukocytes were isolated on 5 dpi. Representative FACS plots ( A ) and absolute number ( B ) of CD3 + T cells were shown, n = 4. Tilapia individuals were i.p. injected with BFA 6 h before sacrifice. Representative FACS plots ( C ) and bar figure ( D ) showing the percentage of IL-2 + T cells in gated CD3 + T cells, n = 3. Histograms showing the levels of CD122 in gated CD3 + T cells ( E ). Tilapia individuals were i.p. injected with BrdU 1 day before sacrifice, and spleen lymphocytes were isolated for assays, and representative FACS plots ( F ) and bar figure ( G ) showing the percentage of BrdU + T cells in gated CD3 + T cells, n = 3–4. Representative FACS plots ( H ) and bar figure ( I ) showing the 7-AAD and Annexin V staining on gated CD3 + T-cell population, n = 5. Tilapia individuals were i.p. injected with BFA 6 h before sacrifice, and spleen lymphocytes were isolated for assays. Representative FACS plots ( J ) and bar figure ( K ) showing the percentage of granzyme B + T cells in gated CD3 + T cells, n = 3. ( L ) A. hydrophila titers in the liver of infected tilapia on 6 dpi, n = 5–6. ( M ) Kaplan–Meier survival plot showing the survival percentage of tilapia, n = 26. Data information: n stands for biological replicates. Error bars indicate mean ± SEM. Significance between the groups was determined by a two-tailed Student’s t test.

Article Snippet: For in vitro activation of AdipoR1, the spleen leukocytes cultured in DMEM (10% FBS, 1% penicillin/streptomycin) were treated with 10 μM AdipoRon (MedChemExpress) for the indicated time.

Techniques: Infection, Injection, Purification, Isolation, Staining, Two Tailed Test

Journal: EMBO Reports

Article Title: CTRP9 engages AdipoR1 and promotes T cell glycolysis and immunity

doi: 10.1038/s44319-025-00640-0

Figure Lengend Snippet: ( A ) Ca 2+ influx of Indo-1–loaded spleen lymphocytes was determined by flow cytometry based on the change in the 450:510 nm ratio after AdipoRon stimulation. ( B , C ) Spleen leukocytes were stimulated with AdipoRon. Western blot assay showing the protein or phosphorylation levels of CaM, CaMKKβ, AMPKα at the indicated times ( B ). Overlaid histograms showing the expression levels of indicated molecules in gated CD3 + T cells at 6 h after stimulation ( C ). ( D ) Spleen leukocytes isolated from tilapia were stimulated with AdipoRon in the presence or absence of CaMKKβ inhibitor STO-609, western blot assay showing the protein or phosphorylation levels of indicated molecules at 6 h after stimulation. ( E ) Tilapia individuals were i.p. injected with or without rOnCTRP9 on days 0 and 1, and the spleen leukocytes were harvested on days 1, 2, and 3 for assay. Western blot showing the protein or phosphorylation levels of CaM, CaMKKβ, AMPKα at indicated times. ( F – R ) Tilapia i.p. injected with CTRP9-specific or control siRNA were infected with A. hydrophila , and spleen leukocytes were harvest on 5 dpi for assay. ( F ) Bar figure showing the percentage of AdipoR1 + T cells in gated lymphocytes, n = 4. ( G ) The absolute number of CD3 + T cells are shown, n = 4. ( H ) Histograms showing the levels of CD122 in gated CD3 + T cells. ( I , J ) Tilapia individuals were i.p. injected with BFA 6 h before sacrifice. Representative FACS plots ( I ) and bar figure ( J ) showing the percentage of IL-2 + T cells in gated CD3 + T cells, n = 3. ( K ) Relative mRNA levels of CD122 , IL-2 , and IFN-γ by qPCR, n = 4. ( L, M ) Representative FACS plots ( L ) and bar figure ( M ) showing the 7-AAD and Annexin V staining on gated CD3 + T-cell population, n = 4. ( N , O ) Tilapia individuals were i.p. injected with BFA 6 h before sacrifice, representative FACS plots ( N ) and bar figure ( O ) showing the percentage of Granzyme B + T cells in gated CD3 + T cells, n = 3. ( P ) Relative mRNA levels of Perforin A and Granzyme B by qPCR, n = 4. ( Q ) A. hydrophila titers in the liver of infected tilapia on 5 dpi, n = 5. ( R ) Kaplan–Meier survival plot showing the survival percentage of tilapia, n = 25. Data information: n stands for biological replicates. Error bars indicate mean ± SEM. Significance between the groups was determined by a two-tailed Student’s t test.

Article Snippet: For in vitro activation of AdipoR1, the spleen leukocytes cultured in DMEM (10% FBS, 1% penicillin/streptomycin) were treated with 10 μM AdipoRon (MedChemExpress) for the indicated time.

Techniques: Flow Cytometry, Western Blot, Phospho-proteomics, Expressing, Isolation, Injection, Control, Infection, Staining, Two Tailed Test

Journal: EMBO Reports

Article Title: CTRP9 engages AdipoR1 and promotes T cell glycolysis and immunity

doi: 10.1038/s44319-025-00640-0

Figure Lengend Snippet: ( A – D ) Tilapia individuals that infected with A. hydrophila were i.p. injected with or without rOnCTRP9 on days 1, 2, and 4, and were i.p. injected with BFA 6 h before sacrifice. Spleen leukocytes were isolated on 5 dpi. Representative FACS plots ( A , C ) and bar figure ( B , D ) showing the percentage of IL-2 + T cells or Granzyme B + T cells in gated CD3 + T cells, n = 3. ( E , F ) Spleen T cells sorted from healthy tilapia were stimulated with mouse anti-tilapia CD3ε and CD28 mAb in the presence or absence of rOnCTRP9 for 6 h, and relative mRNA levels of the indicated molecules were examined by qPCR, n = 4. ( G – J ) Spleen leukocytes that cultured in the medium containing normal glucose (4.5 g/L) and low glucose (1.125 g/L) were treated with rOnCTRP9 or not for 12 h. ( G , H ) Representative FACS plots ( G ) and statistical figure ( H ) showing the 7-AAD and Annexin V staining on gated CD3 + T-cell population, n = 5. ( I , J ) Representative FACS plots ( I ) and bar figure ( J ) showing the percentage of cleaved caspase 8 in gated CD3 + T cells, n = 4. ( K – N ) Spleen leukocytes or sorted T cells were treated with rOnCTRP9 or not. ( K ) Relative mRNA levels of the indicated molecules were examined by qPCR in sorted T cells at 6 h, n = 4. ( L ) Western blot showing the protein levels of the indicated molecules at the indicated time points in leukocytes. ( M ) Glucose uptake in gated CD3 + T cells was measured by 2-NBDG + cells at 5 h after treatment. ( N ) Extracellular acidification rate (ECAR) after 3 h of CTRP9 treatment, n = 6. ( O – Q ) Tilapia individuals infected with A. hydrophila were i.p. injected with or without rat anti-tilapia AdipoR1 antibody on days 1 and 3, or rOnCTRP9 on days 1, 2, and 4. Spleen leukocytes were isolated on 5 dpi. ( O , P ) Histograms ( O ) and bar figure ( P ) showing glucose uptake in gated CD3 + T cells, n = 5. ( Q ) Western blot showing protein levels of the indicated molecules. ( R ) Spleen leukocytes stimulated with rOnCTRP9 were treated with 10 μM Compound C or not for 6 h, and western blot showing the protein levels of Glut1, HK2, and PKM. Data information: n stands for biological replicates. Error bars indicate mean ± SEM. Significance between the groups was determined by a two-tailed Student’s t test.

Article Snippet: For in vitro activation of AdipoR1, the spleen leukocytes cultured in DMEM (10% FBS, 1% penicillin/streptomycin) were treated with 10 μM AdipoRon (MedChemExpress) for the indicated time.

Techniques: Infection, Injection, Isolation, Cell Culture, Staining, Western Blot, Two Tailed Test

Journal: EMBO Reports

Article Title: CTRP9 engages AdipoR1 and promotes T cell glycolysis and immunity

doi: 10.1038/s44319-025-00640-0

Figure Lengend Snippet: ( A – D ) Mouse splenocytes were stimulated with mCTRP9 or mADPN. ( A ) Relative mRNA levels of the indicated molecules were examined by qPCR at 5 h, n = 5. ( B ) Western blot showing protein levels of the indicated molecules at 2 h or 5 h. ( C ) Glucose uptake in gated CD3 + T cells was measured by 2-NBDG staining at 5 h. ( D ) Enzyme activities of HK2 and PFK at 5 h, n = 4. ( E – I ) Mouse splenocytes were stimulated with anti-mouse CD3 plus anti-mouse CD28 in the presence of mADPN. ( E ) Relative mRNA levels of the indicated molecules were examined by qPCR at 5 h, n = 5. ( F ) Western blot showing protein levels of the indicated molecules at 2 h. ( G , H ) Glucose uptake in gated CD4 + or CD8 + T cells was measured by 2-NBDG staining at 5 h ( G ), and a bar figure ( H ) showing the MFI of 2-NBDG in gated CD4 + and CD8 + T cells, n = 5. ( I ) Enzyme activities of HK2 and PFK at 5 h, n = 4. ( J ) Schematic representation of CTRP9 and ADPN differentially regulating T-cell responses by glycolysis and crosstalk between AdipoR1 and T-cell signaling in tilapia and mouse. Data information: The n stands for biological replicates. Error bars indicate mean ± SEM. Significance between the groups was determined by a two-tailed Student’s t test.

Article Snippet: For in vitro activation of AdipoR1, the spleen leukocytes cultured in DMEM (10% FBS, 1% penicillin/streptomycin) were treated with 10 μM AdipoRon (MedChemExpress) for the indicated time.

Techniques: Western Blot, Staining, Two Tailed Test

Journal: American Journal of Translational Research

Article Title: CXCR3 confers sorafenib resistance of HCC cells through regulating metabolic alteration and AMPK pathway

doi:

Figure Lengend Snippet: AMPK pathway is involved in CXCR3-mediated metabolic phenotype switch in SOR-resistance HCC cells. (A) Corresponding network by sequencing analysis based on the different expression genes of Huh7 cells and Huh7-SOR cells indicated AMPK pathway is a critical node during the forming of resistance to SOR. (B, C) Compared to Huh7 cells, p-AMPKα/AMPKα ratio was significantly decreased, levels of adipocytokines including ACSBG1, ADIPOQ, ACSL4 were increased as well as intracellular level of lipid peroxidation product MDA was decreased in Huh7-SOR cells, as shown in above values (D). After CXCR3 knockdown, compared with CXCR3-knockdown parental Huh7 (Huh7-shCXCR3), CXCR3-knockdown Huh7-SOR cells (Huh7-SOR-shCXCR3) didn’t show significant decrease of phosphor-AMPKα/AMPKα ratio and malondialdehyde amount as well as the upregulation of ACSBG1, ADIPOQ, ACSL4 (B-D). All values represent the mean of three independent experiments. *P<0.05 vs Huh7, #P<0.05 vs Huh7-SOR, &P>0.05 vs Huh7-shCXCR3. Moreover, CXCR3-knockdown Huh7-SOR cells were treated with metformin (10 mM) for 24 h, and no significant upregulation of AMPK activity (E, F) and levels of adipocytokines and intracellular MDA (G) were found, compared with those in control (Huh7-SOR-shCXCR3) cells. &P>0.05 vs Huh7-SOR-shCXCR3.

Article Snippet: ADIPOQ (CSB-PA618019YA01HU) and ACSL4 (CSB-PA060488EA01HU) were from CUSABIO, ACSBG1 (GTX16561) was from GeneTex.

Techniques: Sequencing, Expressing, Knockdown, Activity Assay, Control

Journal: Drug Design, Development and Therapy

Article Title: 5-( Bis (3-(2-hydroxyethyl)-1H-indol-2-yl)methyl)-2-hydroxybenzoic acid (BHIMHA): showing a strategy of designing drug to block lung metastasis of tumors

doi: 10.2147/DDDT.S93570

Figure Lengend Snippet: Docking investigation of BHIMHA and staurosporine toward PKCα (PDB: 3IW4). Notes: ( A ) The feature of BHIMHA in the active site of PKCα, and the amplified interactions of energy minimized BHIMHA with the amino acid residues. ( B ) The feature of staurosporine in the active site of PKCα, and the amplified interactions of energy minimized staurosporine with the amino acid residues. Abbreviations: PKC, protein kinase C; BHIMHA, 5-( bis (3-(2-hydroxyethyl)-1H-indol-2-yl)-methyl)-2-hydroxybenzoic acid; staurosporine, (5S,6R,7R,9R)-6-methoxy-5-methyl-7-methylamino-6,7,8,9,15,16-hexahydro-5H,14H-17-oxa-4b,9a,15-triaza-5,9-methanodibenzo[ b, h ]cyclone-na[ jkl ]cyclopenta[ e ]asindacen-14-one; PDB, Protein Data Bank.

Article Snippet: PKCα, PKCβII, PKCγ, PKCη, and PKCδ antibodies were purchased from Boster Biological Engineering Co., Ltd. (Wuhan, People’s Republic of China); PKCθ antibody was purchased from CST (Shanghai) Biological Reagents Company Limited, Shanghai, People’s Republic of China.

Techniques: Amplification

Journal: International Journal of Molecular Sciences

Article Title: Prolonged Chronic Consumption of a High Fat with Sucrose Diet Alters the Morphology of the Small Intestine

doi: 10.3390/ijms22147280

Figure Lengend Snippet: ( A , C ) Immunohistochemistry of adiponectin and adiponectin receptor of proximal tract of the small intestine. (Original magnification: 10×; scale bar: 100 μm). ( B , D ) The expression of adiponectin and its receptor was significantly reduced in HFD w/Suc mice compared to the SD group. * p < 0.05. These images are representative of n = 5 SD, n = 10 HFD w/Suc mice.

Article Snippet: Adiponectin , Boster Biological Technology, Pleasanton, CA, USA; code PA2014-1 , 1:50.

Techniques: Immunohistochemistry, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Prolonged Chronic Consumption of a High Fat with Sucrose Diet Alters the Morphology of the Small Intestine

doi: 10.3390/ijms22147280

Figure Lengend Snippet: Antibodies used with their sources and dilutions.

Article Snippet: Adiponectin , Boster Biological Technology, Pleasanton, CA, USA; code PA2014-1 , 1:50.

Techniques:

Journal: Arthritis Research & Therapy

Article Title: Potential therapeutic antibodies targeting specific adiponectin isoforms in rheumatoid arthritis

doi: 10.1186/s13075-018-1736-3

Figure Lengend Snippet: Detection of serum adiponectin isoforms with monoclonal antibodies (mAbs) against recombinant human adiponectin. a Western blot to screen mAbs detecting different adiponectin isoforms. Human serum was loaded into each lane of the gel and resolved by polyacrylamide gel electrophoresis (PAGE) and then transferred to a membrane. Each lane of the membrane was then separately cut and incubated with each mAb from hybridoma culture supernatant. After the secondary antibody was probed, the cut membrane was combined to form one sheet and developed with ECL solution. Eleven different mAbs from culture supernatant were tested to investigate their recognition patterns of adiponectin isoforms in serum. The mAb KH7–33, in lane 3, was selected as a representative recognizing only the middle molecular weight (MMW) isoform of adiponectin. The mAb KH7–41, in lane 4, was selected as a representative recognizing both the MMW and low molecular weight (LMW) isoforms of adiponectin. The mAb KH4–8, in lane 8, was selected as a representative mAb recognizing both the MMW and HMW isoforms of adiponectin. b Comparison of the mAb recognition patterns of adiponectin isoforms in human, mouse, and rat sera by Western blot. Human, mouse, and rat sera were separated by sodium dodecyl sulfate–PAGE. All three mAbs (KH7–33, KH7–41, and KH4–8) recognized the rat and mouse adiponectin MMW isoform. Lane 1, KH7–41; lane 2, KH7–33; lane 3, KH4–8; lane 4, commercial antibody against human adiponectin (Boster Immunoleader cat. no. PB9001) or mouse/rat adiponectin (Boster Immunoleader cat. no. PB9011). Mouse and rat serum were obtained via heart puncture of male BALB/c mice and Sprague Dawley rats (8 weeks old), respectively. Human serum was obtained from a male volunteer (55 years old)

Article Snippet: Lane 1, KH7–41; lane 2, KH7–33; lane 3, KH4–8; lane 4, commercial antibody against human adiponectin (Boster Immunoleader cat. no. PB9001) or mouse/rat adiponectin (Boster Immunoleader cat. no. PB9011).

Techniques: Bioprocessing, Recombinant, Western Blot, Polyacrylamide Gel Electrophoresis, Membrane, Incubation, Molecular Weight, Comparison

Journal: Arthritis Research & Therapy

Article Title: Potential therapeutic antibodies targeting specific adiponectin isoforms in rheumatoid arthritis

doi: 10.1186/s13075-018-1736-3

Figure Lengend Snippet: Immunohistological assays in human tissues with monoclonal antibodies (mAbs). To determine the pattern of recognition of adiponectin isoforms in human tissues by mAbs, a normal human adipose tissue was immunostained with mAbs (KH7–41, KH7–33, and KH4–8) (200×, scale bar = 25 μm). mAbs recognized adiponectin in the nucleus of adipocytes (blue arrow) and in vessels and endothelial cells (red arrow). b Human lung, kidney, and pancreas were stained with the KH7–41, KH7–33, and KH4–8 mAbs (100×). Abbreviations: HMW high molecular weight, LMW low molecular weight, MMW middle molecular weight

Article Snippet: Lane 1, KH7–41; lane 2, KH7–33; lane 3, KH4–8; lane 4, commercial antibody against human adiponectin (Boster Immunoleader cat. no. PB9001) or mouse/rat adiponectin (Boster Immunoleader cat. no. PB9011).

Techniques: Bioprocessing, Staining, High Molecular Weight, Molecular Weight

Journal: Arthritis Research & Therapy

Article Title: Potential therapeutic antibodies targeting specific adiponectin isoforms in rheumatoid arthritis

doi: 10.1186/s13075-018-1736-3

Figure Lengend Snippet: Inhibition of adiponectin-mediated gene expression in vitro by monoclonal antibodies (mAbs). To test the ability of the mAb KH4–8 to block adiponectin function, ( a ) human osteoblasts and ( b ) human umbilical vein endothelial cells (HUVECs) were treated with adiponectin (ADIPO) or KH4–8 mAb (mAb) or both. The mAb (~120 μg/mL) and recombinant adiponectin (2.5 μg/mL) were mixed and incubated for 1 h before being used to treat cells. After 24-h treatment, the culture supernatants were collected and frozen, and interleukin-6 (IL-6) and IL-8 were measured by using enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN, USA). The experiments were performed in quadruplicate. The data shown are representative of three independent experiments, and similar results were obtained with all three mAbs. Values are expressed as mean ± standard error of the mean. The expression levels of the factors were compared between groups by using the Mann–Whitney test. * P <0.05, ** P <0.01 versus the untreated group, # P <0.05, ## P <0.01 versus the group treated with adiponectin and mAb

Article Snippet: Lane 1, KH7–41; lane 2, KH7–33; lane 3, KH4–8; lane 4, commercial antibody against human adiponectin (Boster Immunoleader cat. no. PB9001) or mouse/rat adiponectin (Boster Immunoleader cat. no. PB9011).

Techniques: Inhibition, Gene Expression, In Vitro, Bioprocessing, Blocking Assay, Recombinant, Incubation, Enzyme-linked Immunosorbent Assay, Expressing, MANN-WHITNEY

Journal: Arthritis Research & Therapy

Article Title: Potential therapeutic antibodies targeting specific adiponectin isoforms in rheumatoid arthritis

doi: 10.1186/s13075-018-1736-3

Figure Lengend Snippet: Epitope mapping of monoclonal antibody (mAb) KH4–8 against adiponectin. To identify the epitope-recognizing site of the KH4–8 mAb, PEPperMAP ® technology was performed as described in the Methods. Human adiponectin was translated into linear 15–amino acid peptides with a peptide-peptide overlap of 14 amino acids. Human adiponectin peptide microarrays were incubated with mouse mAb KH4–8 at different concentrations followed by staining with secondary goat anti-mouse IgG (H + L) DyLight680 antibody. The light intensity was read by a reader. The amino acid sequence QQNHYD (139–144) was confirmed from among the full 244–amino acid sequence to be the epitope of adiponectin

Article Snippet: Lane 1, KH7–41; lane 2, KH7–33; lane 3, KH4–8; lane 4, commercial antibody against human adiponectin (Boster Immunoleader cat. no. PB9001) or mouse/rat adiponectin (Boster Immunoleader cat. no. PB9011).

Techniques: Incubation, Staining, Sequencing

Journal: Arthritis Research & Therapy

Article Title: Potential therapeutic antibodies targeting specific adiponectin isoforms in rheumatoid arthritis

doi: 10.1186/s13075-018-1736-3

Figure Lengend Snippet: Anti-inflammatory effect of monoclonal antibodies (mAbs) KH4–8 and KH7–33 on the expression of serum pro-inflammatory cytokines of the collagen-induced arthritis mouse model. The serum levels of adiponectin, interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and receptor activator of nuclear factor-kappa Β ligand (RANKL) were analyzed by using the Luminex system. Values are expressed as mean ± standard error of the mean. The expression levels of the factors were compared between groups ( n = 8) by using the Mann–Whitney test. ** P <0.01, * P <0.05 versus normal (NOR) group, and ## P <0.01, ## P <0.05 versus the control (CON) group. Abbreviations: ns not significant, pre prednisolone

Article Snippet: Lane 1, KH7–41; lane 2, KH7–33; lane 3, KH4–8; lane 4, commercial antibody against human adiponectin (Boster Immunoleader cat. no. PB9001) or mouse/rat adiponectin (Boster Immunoleader cat. no. PB9011).

Techniques: Bioprocessing, Expressing, Luminex, MANN-WHITNEY, Control

Journal: Arthritis Research & Therapy

Article Title: Potential therapeutic antibodies targeting specific adiponectin isoforms in rheumatoid arthritis

doi: 10.1186/s13075-018-1736-3

Figure Lengend Snippet: Anti-adiponectin antibodies reduce the histological signs of inflammation. The upper and lower panels present hematoxylin and eosin (H&E) staining and immunostaining against mouse adiponectin of mouse knee joints (n = 8), respectively. a Normal, b control, saline-treated arthritic, c KH7–33-treated arthritic, d KH4–8-treated arthritic, and ( e ) prednisolone-treated arthritic mice. Tissue structure was visualized by using H&E staining (original magnification, 40×). Scale bar = 2 mm. f Arthritic symptoms were evaluated by scoring the degree of inflammation on H&E histological sections of knee joints as described in the Methods. Small blue squares on H&E staining are magnified in the upper right corner (400×). Abbreviations: C cartilage, F femur, M meniscus, S subchondral bone, T tibia. In the lower panel, immunohistochemistry (IHC) reveals adiponectin expression in collagen-induced arthritis mouse joints (200×). The increased adiponectin expression level observed on IHC was not decreased by monoclonal antibody treatment. Adiponectin immunostaining score level was evaluated as described in the Methods. Results are presented as the mean of experiments (± standard error of the mean indicated by error bar) (one-way analysis of variance followed by Dunn’s multiple comparison test). *** P <0.001 versus the normal (NOR) group and # P <0.05, ## P <0.01 versus the control (CON) group. Abbreviation: pre prednisolone

Article Snippet: Lane 1, KH7–41; lane 2, KH7–33; lane 3, KH4–8; lane 4, commercial antibody against human adiponectin (Boster Immunoleader cat. no. PB9001) or mouse/rat adiponectin (Boster Immunoleader cat. no. PB9011).

Techniques: Staining, Immunostaining, Control, Saline, Immunohistochemistry, Expressing, Comparison

Journal: Arthritis Research & Therapy

Article Title: Potential therapeutic antibodies targeting specific adiponectin isoforms in rheumatoid arthritis

doi: 10.1186/s13075-018-1736-3

Figure Lengend Snippet: Detection of serum adiponectin isoforms with monoclonal antibodies (mAbs) against recombinant human adiponectin. a Western blot to screen mAbs detecting different adiponectin isoforms. Human serum was loaded into each lane of the gel and resolved by polyacrylamide gel electrophoresis (PAGE) and then transferred to a membrane. Each lane of the membrane was then separately cut and incubated with each mAb from hybridoma culture supernatant. After the secondary antibody was probed, the cut membrane was combined to form one sheet and developed with ECL solution. Eleven different mAbs from culture supernatant were tested to investigate their recognition patterns of adiponectin isoforms in serum. The mAb KH7–33, in lane 3, was selected as a representative recognizing only the middle molecular weight (MMW) isoform of adiponectin. The mAb KH7–41, in lane 4, was selected as a representative recognizing both the MMW and low molecular weight (LMW) isoforms of adiponectin. The mAb KH4–8, in lane 8, was selected as a representative mAb recognizing both the MMW and HMW isoforms of adiponectin. b Comparison of the mAb recognition patterns of adiponectin isoforms in human, mouse, and rat sera by Western blot. Human, mouse, and rat sera were separated by sodium dodecyl sulfate–PAGE. All three mAbs (KH7–33, KH7–41, and KH4–8) recognized the rat and mouse adiponectin MMW isoform. Lane 1, KH7–41; lane 2, KH7–33; lane 3, KH4–8; lane 4, commercial antibody against human adiponectin (Boster Immunoleader cat. no. PB9001) or mouse/rat adiponectin (Boster Immunoleader cat. no. PB9011). Mouse and rat serum were obtained via heart puncture of male BALB/c mice and Sprague Dawley rats (8 weeks old), respectively. Human serum was obtained from a male volunteer (55 years old)

Article Snippet: Lane 1, KH7–41; lane 2, KH7–33; lane 3, KH4–8; lane 4, commercial antibody against human adiponectin (Boster Immunoleader cat. no. PB9001) or mouse/rat adiponectin (Boster Immunoleader cat. no. PB9011).

Techniques: Bioprocessing, Recombinant, Western Blot, Polyacrylamide Gel Electrophoresis, Membrane, Incubation, Molecular Weight, Comparison

Journal: Arthritis Research & Therapy

Article Title: Potential therapeutic antibodies targeting specific adiponectin isoforms in rheumatoid arthritis

doi: 10.1186/s13075-018-1736-3

Figure Lengend Snippet: Immunohistological assays in human tissues with monoclonal antibodies (mAbs). To determine the pattern of recognition of adiponectin isoforms in human tissues by mAbs, a normal human adipose tissue was immunostained with mAbs (KH7–41, KH7–33, and KH4–8) (200×, scale bar = 25 μm). mAbs recognized adiponectin in the nucleus of adipocytes (blue arrow) and in vessels and endothelial cells (red arrow). b Human lung, kidney, and pancreas were stained with the KH7–41, KH7–33, and KH4–8 mAbs (100×). Abbreviations: HMW high molecular weight, LMW low molecular weight, MMW middle molecular weight

Article Snippet: Lane 1, KH7–41; lane 2, KH7–33; lane 3, KH4–8; lane 4, commercial antibody against human adiponectin (Boster Immunoleader cat. no. PB9001) or mouse/rat adiponectin (Boster Immunoleader cat. no. PB9011).

Techniques: Bioprocessing, Staining, High Molecular Weight, Molecular Weight

Journal: Arthritis Research & Therapy

Article Title: Potential therapeutic antibodies targeting specific adiponectin isoforms in rheumatoid arthritis

doi: 10.1186/s13075-018-1736-3

Figure Lengend Snippet: Inhibition of adiponectin-mediated gene expression in vitro by monoclonal antibodies (mAbs). To test the ability of the mAb KH4–8 to block adiponectin function, ( a ) human osteoblasts and ( b ) human umbilical vein endothelial cells (HUVECs) were treated with adiponectin (ADIPO) or KH4–8 mAb (mAb) or both. The mAb (~120 μg/mL) and recombinant adiponectin (2.5 μg/mL) were mixed and incubated for 1 h before being used to treat cells. After 24-h treatment, the culture supernatants were collected and frozen, and interleukin-6 (IL-6) and IL-8 were measured by using enzyme-linked immunosorbent assay (ELISA) (R&D Systems, Minneapolis, MN, USA). The experiments were performed in quadruplicate. The data shown are representative of three independent experiments, and similar results were obtained with all three mAbs. Values are expressed as mean ± standard error of the mean. The expression levels of the factors were compared between groups by using the Mann–Whitney test. * P <0.05, ** P <0.01 versus the untreated group, # P <0.05, ## P <0.01 versus the group treated with adiponectin and mAb

Article Snippet: Lane 1, KH7–41; lane 2, KH7–33; lane 3, KH4–8; lane 4, commercial antibody against human adiponectin (Boster Immunoleader cat. no. PB9001) or mouse/rat adiponectin (Boster Immunoleader cat. no. PB9011).

Techniques: Inhibition, Gene Expression, In Vitro, Bioprocessing, Blocking Assay, Recombinant, Incubation, Enzyme-linked Immunosorbent Assay, Expressing, MANN-WHITNEY

Journal: Arthritis Research & Therapy

Article Title: Potential therapeutic antibodies targeting specific adiponectin isoforms in rheumatoid arthritis

doi: 10.1186/s13075-018-1736-3

Figure Lengend Snippet: Epitope mapping of monoclonal antibody (mAb) KH4–8 against adiponectin. To identify the epitope-recognizing site of the KH4–8 mAb, PEPperMAP ® technology was performed as described in the Methods. Human adiponectin was translated into linear 15–amino acid peptides with a peptide-peptide overlap of 14 amino acids. Human adiponectin peptide microarrays were incubated with mouse mAb KH4–8 at different concentrations followed by staining with secondary goat anti-mouse IgG (H + L) DyLight680 antibody. The light intensity was read by a reader. The amino acid sequence QQNHYD (139–144) was confirmed from among the full 244–amino acid sequence to be the epitope of adiponectin

Article Snippet: Lane 1, KH7–41; lane 2, KH7–33; lane 3, KH4–8; lane 4, commercial antibody against human adiponectin (Boster Immunoleader cat. no. PB9001) or mouse/rat adiponectin (Boster Immunoleader cat. no. PB9011).

Techniques: Incubation, Staining, Sequencing

Journal: Arthritis Research & Therapy

Article Title: Potential therapeutic antibodies targeting specific adiponectin isoforms in rheumatoid arthritis

doi: 10.1186/s13075-018-1736-3

Figure Lengend Snippet: Anti-inflammatory effect of monoclonal antibodies (mAbs) KH4–8 and KH7–33 on the expression of serum pro-inflammatory cytokines of the collagen-induced arthritis mouse model. The serum levels of adiponectin, interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and receptor activator of nuclear factor-kappa Β ligand (RANKL) were analyzed by using the Luminex system. Values are expressed as mean ± standard error of the mean. The expression levels of the factors were compared between groups ( n = 8) by using the Mann–Whitney test. ** P <0.01, * P <0.05 versus normal (NOR) group, and ## P <0.01, ## P <0.05 versus the control (CON) group. Abbreviations: ns not significant, pre prednisolone

Article Snippet: Lane 1, KH7–41; lane 2, KH7–33; lane 3, KH4–8; lane 4, commercial antibody against human adiponectin (Boster Immunoleader cat. no. PB9001) or mouse/rat adiponectin (Boster Immunoleader cat. no. PB9011).

Techniques: Bioprocessing, Expressing, Luminex, MANN-WHITNEY, Control

Journal: Arthritis Research & Therapy

Article Title: Potential therapeutic antibodies targeting specific adiponectin isoforms in rheumatoid arthritis

doi: 10.1186/s13075-018-1736-3

Figure Lengend Snippet: Anti-adiponectin antibodies reduce the histological signs of inflammation. The upper and lower panels present hematoxylin and eosin (H&E) staining and immunostaining against mouse adiponectin of mouse knee joints (n = 8), respectively. a Normal, b control, saline-treated arthritic, c KH7–33-treated arthritic, d KH4–8-treated arthritic, and ( e ) prednisolone-treated arthritic mice. Tissue structure was visualized by using H&E staining (original magnification, 40×). Scale bar = 2 mm. f Arthritic symptoms were evaluated by scoring the degree of inflammation on H&E histological sections of knee joints as described in the Methods. Small blue squares on H&E staining are magnified in the upper right corner (400×). Abbreviations: C cartilage, F femur, M meniscus, S subchondral bone, T tibia. In the lower panel, immunohistochemistry (IHC) reveals adiponectin expression in collagen-induced arthritis mouse joints (200×). The increased adiponectin expression level observed on IHC was not decreased by monoclonal antibody treatment. Adiponectin immunostaining score level was evaluated as described in the Methods. Results are presented as the mean of experiments (± standard error of the mean indicated by error bar) (one-way analysis of variance followed by Dunn’s multiple comparison test). *** P <0.001 versus the normal (NOR) group and # P <0.05, ## P <0.01 versus the control (CON) group. Abbreviation: pre prednisolone

Article Snippet: Lane 1, KH7–41; lane 2, KH7–33; lane 3, KH4–8; lane 4, commercial antibody against human adiponectin (Boster Immunoleader cat. no. PB9001) or mouse/rat adiponectin (Boster Immunoleader cat. no. PB9011).

Techniques: Staining, Immunostaining, Control, Saline, Immunohistochemistry, Expressing, Comparison