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