Journal: Cancers

Article Title: Visfatin Mediates Malignant Behaviors through Adipose-Derived Stem Cells Intermediary in Breast Cancer

doi: 10.3390/cancers12010029

Figure Lengend Snippet: GDF15 play a crucial role in the regulations of breast cancer cells activities by visfatin-treated ADSCs (vADSCs). ( A ) After the three-day co-culture of MDA-MB-231 cells and vADSCs (V50 and V100 group) or untreated ADSCs (Ctrl group), the CM was collected and analyzed by using a cytokine array kit. ( B ) The expression of GDF15 in the co-cultured CM was validated by ELISA represented in a histogram. The ADSCs and MDA-MB-231 cells collected from the co-culture system were extracted for cell lysate to analyze the GDF15 expression by western blotting. ( C ) The migration and invasion of MDA-MB-231 treated with GDF15 at various concentrations for 48 h were evaluated by using a transwell system. ( D ) The indirect co-culture was performed in the presence or absence of the GDF15 neutralizing antibody of for three days. After that, the migration and invasion of the MDA-MB-231 cells collected from the co-culture were evaluated in a transwell system. ( E ) The expression of phosphor-AKT (pAKT) of MDA-MB-231 treated with GDF15 (50 ng/mL) at different time point was detected by western blotting. ( F ) The pAKT was detected in the MDA-MB-231 cells from the co-culture by western blotting. ( G ) After the three-day co-culture in the presence or absence of the wortmannin (400 nM), the MDA-MB-231 cells were collected from the co-culture for performing the migration assay. All experiments were performed in triplicate.

Article Snippet: For performing cell invasion or migration assay, the MDA-MB-231 cells collected from the co-culture system or treated with human recombinant GDF15 (10936-H01H, Sino Biological, Wayne, PA, USA) were suspended in serum-free DMEM and transferred to the 24-well transwell inserts coated with or without Matrigel, respectively (8 μm pores, Corning).

Techniques: Co-Culture Assay, Expressing, Cell Culture, Enzyme-linked Immunosorbent Assay, Western Blot, Migration

Journal: Cancers

Article Title: Visfatin Mediates Malignant Behaviors through Adipose-Derived Stem Cells Intermediary in Breast Cancer

doi: 10.3390/cancers12010029

Figure Lengend Snippet: Visfatin-primed ADSCs promoted the tube formation of HUVEC. ( A ) HUVEC cells were co-cultured with visfatin-treated ADSCs or untreated ADSCs, noted as V50 and V100 or Ctrl, respectively, for three days. The HUVEC cells were collected from the co-culture and seeded in a matrix gel-coated 96-well plate. The tube formation of HUVEC was observed using a microscope. The length of branches was determined by using the ImageJ software. ( B ) After the three-day co-culture in the presence or absence of GDF15 neutralizing antibody (GDF15 Nab, 5 μg/mL), the HUVEC cells were collected from the co-culture for performing the tube formation assay. The experiments were performed in triplicate.

Article Snippet: For performing cell invasion or migration assay, the MDA-MB-231 cells collected from the co-culture system or treated with human recombinant GDF15 (10936-H01H, Sino Biological, Wayne, PA, USA) were suspended in serum-free DMEM and transferred to the 24-well transwell inserts coated with or without Matrigel, respectively (8 μm pores, Corning).

Techniques: Cell Culture, Co-Culture Assay, Microscopy, Software, Tube Formation Assay

Journal: Cancers

Article Title: Visfatin Mediates Malignant Behaviors through Adipose-Derived Stem Cells Intermediary in Breast Cancer

doi: 10.3390/cancers12010029

Figure Lengend Snippet: Visfatin-pretreated ADSCs (vADSCs) enhanced the tumor growth and metastasis in human breast cancer xenograft mouse model. ( A ) The nude mice were injected with mixture of MDA-MB-231 and untreated ADSCs (uADSCs) or vADSCs, noted as Ctrl or V50, respectively, to the mammary fat pads. The tumor volumes were measured every week after injection (Ctrl, n = 5; V50, n = 6). ( B ) After sacrificing the mice, the weight of the resected tumor was measured. ( C ) The expressions of GDF15, β-catenin, CD31, and pAKT in the tumor sections were detected by immunohistochemistry. The IHC score was calculated by multiplying the percentage of positive cells by the intensity and present as histogram. ( D ) The luciferase-expressing MDA-MB-231 were collected and injected into the tail vein of NOD/SCID mice after co-culturing with uADSCs or vADSCs, noted as Ctrl or V50, respectively (Ctrl, n = 8; V50, n = 8). The IVIS radiance signals of the mice were assessed at week 4. The representative images of high and low signal were shown. The statistical differences were calculated by t-test, *, p -value < 0.05; **, p -value < 0.01.

Article Snippet: For performing cell invasion or migration assay, the MDA-MB-231 cells collected from the co-culture system or treated with human recombinant GDF15 (10936-H01H, Sino Biological, Wayne, PA, USA) were suspended in serum-free DMEM and transferred to the 24-well transwell inserts coated with or without Matrigel, respectively (8 μm pores, Corning).

Techniques: Injection, Immunohistochemistry, Luciferase, Expressing

Journal: Cancers

Article Title: Visfatin Mediates Malignant Behaviors through Adipose-Derived Stem Cells Intermediary in Breast Cancer

doi: 10.3390/cancers12010029

Figure Lengend Snippet: The expressions of visfatin, GDF15, and pAKT in the specimens from breast cancer patients. ( A ) The expressions of visfatin, GDF15, and pAKT in breast cancer tissue microarray (n = 96) were detected by immunohistochemistry. The representative images of high expression levels (No. 1) and low expression levels (No. 2) were shown. The IHC score was calculated by multiplying the percentage of positive cells by the intensity, which was identified using HistoQuest Analysis Software. ( B ) The correlations between visfatin, GDF15, and pAKT according to the IHC score were calculated by using the online Pearson correlation coefficient calculator. ( C ) The correlation of serum levels of GDF15 and visfatin of breast cancer patients (n = 120) determined by ELISA was also calculated by using the online Pearson correlation coefficient calculator.

Article Snippet: For performing cell invasion or migration assay, the MDA-MB-231 cells collected from the co-culture system or treated with human recombinant GDF15 (10936-H01H, Sino Biological, Wayne, PA, USA) were suspended in serum-free DMEM and transferred to the 24-well transwell inserts coated with or without Matrigel, respectively (8 μm pores, Corning).

Techniques: Microarray, Immunohistochemistry, Expressing, Software, Enzyme-linked Immunosorbent Assay

Journal: Cancers

Article Title: Visfatin Mediates Malignant Behaviors through Adipose-Derived Stem Cells Intermediary in Breast Cancer

doi: 10.3390/cancers12010029

Figure Lengend Snippet: Visfatin mediates its effects both directly via cAbl/STAT3 and indirectly mediated by ADSCs via GDF15/AKT on promoting malignant behavior in breast cancer. Previously, we discovered visfatin mainly produced by adipocytes promoted breast cancer cells directly through activation of c-Abl and STAT3, which was blocked by Imatinib and Stattic inhibitor, respectively (black arrow). In this study, we showed that visfatin can act via an indirect pathway by priming ADSCs, which may be recruited from the adipose tissue to tumor site or generated from autologous fat transfer, to produce GDF15 that stimulated AKT activation in breast cancer cells to promote malignant behaviors (white arrow). The effect can be blocked by the treatment of GDF15 neutralizing Ab or Wortmannin inhibitor.

Article Snippet: For performing cell invasion or migration assay, the MDA-MB-231 cells collected from the co-culture system or treated with human recombinant GDF15 (10936-H01H, Sino Biological, Wayne, PA, USA) were suspended in serum-free DMEM and transferred to the 24-well transwell inserts coated with or without Matrigel, respectively (8 μm pores, Corning).

Techniques: Produced, Activation Assay, Generated

Journal: Redox Biology

Article Title: GDF15 nanotherapy ameliorates NLRP3-associated redox imbalance and cardiac injury in sepsis

doi: 10.1016/j.redox.2025.103897

Figure Lengend Snippet: Multifaceted validation of GDF15 changes in serum from SICM patients and their clinical associations. (A) GDF15 levels were quantified using the Luminex platform. (B) A volcano plot illustrated the gene expression distribution of GDF15 among differentially expressed genes (DEGs) in whole blood. (C) A heatmap displayed the expression profiles of GDF15 and inflammatory cytokines. (D) Serum GDF15 levels in patients. (E) Pearson correlation analysis demonstrated the association between GDF15 and SOFA score, as well as EF. (F) ROC curves were plotted to assess the diagnostic accuracy of GDF15 and SOFA score in identifying SICM. (G) Multivariate logistic regression analysis was performed to identify independent risk factors for the development of SICM in septic patients. ∗p < 0.05 indicates significant differences; ns: no significant differences.

Article Snippet: Enzyme-linked immunosorbent assays (ELISA) were conducted using commercial kits to quantify serum levels of human GDF15 (Proteintech, Cat# KE00108), mouse GDF15 (Proteintech, Cat# KE10082), and mouse IL-6 (MULTI SCIENCES, Cat# EK206).

Techniques: Biomarker Discovery, Luminex, Gene Expression, Expressing, Diagnostic Assay

Journal: Redox Biology

Article Title: GDF15 nanotherapy ameliorates NLRP3-associated redox imbalance and cardiac injury in sepsis

doi: 10.1016/j.redox.2025.103897

Figure Lengend Snippet: Upregulation of GDF15 in the SICM model. (A) A schematic workflow for the establishment of the SICM model in C57BL/6J mice via intraperitoneal injection of LPS or saline. (B) Cardiac contractile function parameters, including EF and FS. (C) Serum levels of GDF15 and IL-6. (D) Histopathological analysis of heart tissue, H&E staining (left) and immunohistochemical staining for Ly6G and CD68 (right). Black arrows indicate inflammatory cell infiltration; scale bar: 50 μm. (E) Western blot analysis of GDF15 protein expression in heart tissue. n = 4. (F) qPCR analysis of Gdf15 , Bnp , Il-1β , Il-6 , Icam-1 and Vcam- 1 mRNA levels in heart tissue. (G) Identification of GDF15-positive cells in single-cell RNA-sequencing dataset ( GSE190856 ). (H) qPCR analysis of Gdf15 and Il-1β , Il-6, Nos2, Ptgs2 mRNA expression in BMDM after LPS stimulation. ∗p < 0.05 indicates significant differences; n = 6 per group.

Article Snippet: Enzyme-linked immunosorbent assays (ELISA) were conducted using commercial kits to quantify serum levels of human GDF15 (Proteintech, Cat# KE00108), mouse GDF15 (Proteintech, Cat# KE10082), and mouse IL-6 (MULTI SCIENCES, Cat# EK206).

Techniques: Injection, Saline, Staining, Immunohistochemical staining, Western Blot, Expressing, RNA Sequencing

Journal: Redox Biology

Article Title: GDF15 nanotherapy ameliorates NLRP3-associated redox imbalance and cardiac injury in sepsis

doi: 10.1016/j.redox.2025.103897

Figure Lengend Snippet: GDF15 deficiency exacerbates LPS-induced SICM in mice. (A) Schematic workflow for the establishment of the SICM model in Gdf15 −/− mice. Gdf15 −/− mice were intraperitoneally injected with LPS or saline to induce SICM, with tissue samples collected 24 h post-injection for further analysis. (B) Echocardiographic assessment of EF and FS. (C) H&E staining of heart tissue, black arrows indicate inflammatory cell infiltration. scale bar: 50 μm. (D) CD68 immunofluorescence staining of heart tissue. Blue staining highlights nuclei, red staining identifies CD68 + macrophages; scale bar: 20 μm. (E) qPCR analysis of mRNA expression levels of Bnp , Il-1β, Il-6, and Mcp-1 in heart tissue. n = 6 per group.

Article Snippet: Enzyme-linked immunosorbent assays (ELISA) were conducted using commercial kits to quantify serum levels of human GDF15 (Proteintech, Cat# KE00108), mouse GDF15 (Proteintech, Cat# KE10082), and mouse IL-6 (MULTI SCIENCES, Cat# EK206).

Techniques: Injection, Saline, Staining, Immunofluorescence, Expressing

Journal: Redox Biology

Article Title: GDF15 nanotherapy ameliorates NLRP3-associated redox imbalance and cardiac injury in sepsis

doi: 10.1016/j.redox.2025.103897

Figure Lengend Snippet: MGP exerts anti-inflammatory effects via the MYPT1/AKT/YBX-1 signaling pathway. (A) IP-MS of BMDM to identify the interaction with GDF15. MYPT1 is marked in red. (B) Z-DOCK predicted the interaction domain between GDF15 and MYPT1. Pink represents GDF15, green represents MYPT1, and the boxed region indicates the binding domain. (C) Co-IP combined with Western blot analysis of GDF-15 and MYPT1 binding in macrophages after LPS treatment. (n = 3). (D) Immunofluorescence detection of co-localization between GDF15 (green) and MYPT1 (red), with blue staining for nuclei. Scale bar: 20 μm. (E) Protein expression levels of p -YBX-1, YBX-1, and p -AKT, AKT in BMDM after LPS and/or MGP treatment, with gray-scale intensity analysis of relative expression differences. (F) Representative immunofluorescence images of YBX-1 staining in BMDM after LPS and/or MGP treatment. Blue staining highlights nuclei, and red staining identifies YBX-1. Scale bar: 20 μm ∗p < 0.05, significantly different from control group. #p < 0.05, significantly different from LPS group. n = 6 per group.

Article Snippet: Enzyme-linked immunosorbent assays (ELISA) were conducted using commercial kits to quantify serum levels of human GDF15 (Proteintech, Cat# KE00108), mouse GDF15 (Proteintech, Cat# KE10082), and mouse IL-6 (MULTI SCIENCES, Cat# EK206).

Techniques: Protein-Protein interactions, Binding Assay, Co-Immunoprecipitation Assay, Western Blot, Immunofluorescence, Staining, Expressing, Control

Journal: Redox Biology

Article Title: GDF15 nanotherapy ameliorates NLRP3-associated redox imbalance and cardiac injury in sepsis

doi: 10.1016/j.redox.2025.103897

Figure Lengend Snippet: YBX-1 mediates GDF15-mediated transcriptional regulation of the NLRP3 pathway. (A) qPCR analysis of mRNA expression levels of Nlrp3, Asc , and Il-1β in LPS-stimulated BMDM after Si- Ybx-1 . (B) Western blot analysis of protein expression levels of NLRP3 and IL-1β in LPS-stimulated BMDM after YBX-1 knockdown. (C) qPCR analysis of mRNA expression levels of Nlrp3 and Il-1β in LPS and MGP-treated BMDM after YBX-1 knockdown. (D) Schematic diagram of the luciferase reporter plasmid for the Nlrp3 promoter. (E) Luciferase activity of pcDNA3.1-YBX-1 or empty vector-transfected cells. (F) Luciferase activity after LPS and MGP treatment. n = 6 per group.

Article Snippet: Enzyme-linked immunosorbent assays (ELISA) were conducted using commercial kits to quantify serum levels of human GDF15 (Proteintech, Cat# KE00108), mouse GDF15 (Proteintech, Cat# KE10082), and mouse IL-6 (MULTI SCIENCES, Cat# EK206).

Techniques: Expressing, Western Blot, Knockdown, Luciferase, Plasmid Preparation, Activity Assay, Transfection

Journal: Redox Biology

Article Title: GDF15 nanotherapy ameliorates NLRP3-associated redox imbalance and cardiac injury in sepsis

doi: 10.1016/j.redox.2025.103897

Figure Lengend Snippet: Mechanism of action of macrophage-biomimetic nanocarriers delivering GDF15 to target the YBX-1-NLRP3 axis in SICM. Macrophage-biomimetic nanocarriers loaded with rhGDF15 are targeted to inflammatory sites in the heart, enhancing local drug accumulation, while GDF15 binds to MYPT1 to inhibit YBX-1 phosphorylation and block its nuclear translocation, leading to reduced nuclear YBX-1 expression and decreased transcriptional activity of the Nlrp3 promoter, which suppresses NLRP3 inflammasome assembly and pro-inflammatory cytokine release such as IL-1β, ultimately alleviating macrophage inflammatory responses, myocardial cell injury, and improving cardiac function in SICM.

Article Snippet: Enzyme-linked immunosorbent assays (ELISA) were conducted using commercial kits to quantify serum levels of human GDF15 (Proteintech, Cat# KE00108), mouse GDF15 (Proteintech, Cat# KE10082), and mouse IL-6 (MULTI SCIENCES, Cat# EK206).

Techniques: Phospho-proteomics, Blocking Assay, Translocation Assay, Expressing, Activity Assay

Journal: Frontiers in Immunology

Article Title: Growth/Differentiation Factor-15 (GDF-15): From Biomarker to Novel Targetable Immune Checkpoint

doi: 10.3389/fimmu.2020.00951

Figure Lengend Snippet: Correlation between GDF-15 serum and/or tumor levels with clinical outcome in different cancer types.

Article Snippet: , University Hospital Malmo, Sweden , 138 multiple myeloma patients , GDF-15 MILLIPLEX MAP Human Panel (Millipore) , Serum , 1,008 pg/mL , , GDF-15 above threshold correlates with shorter OS , ( ) .

Techniques: Clinical Proteomics, Serum Assay, Expressing, Enzyme-linked Immunosorbent Assay, Biomarker Discovery, Affinity Chromatography, Purification, Immunostaining

Journal: The Journal of Experimental Medicine

Article Title: Growth differentiation factor 15 deficiency protects against atherosclerosis by attenuating CCR2-mediated macrophage chemotaxis

doi: 10.1084/jem.20100370

Figure Lengend Snippet: Effects of GDF-15 deficiency on plaque stability. (A) Necrotic core size depicted as percentage among total plaque area. (B) Cellular apoptosis was visualized by TUNEL staining and depicted as TUNEL + cells among all mononuclear cells (including representative pictures) in week-12 plaques. *, P < 0.05, compared with WT controls ( n = 9 per group). Arrows indicate TUNEL-positive nuclei. (C) S/G2 phase arrest (depicted as percentage among total cells) in RAW 264.7 macrophages after treatment with 10 ng/ml GDF-15 (gray bars) and 100 ng/ml α-TGFβRII (black bars). **, P < 0.01; ***, P < 0.001, compared with untreated controls (white bars); #, P < 0.001, compared with GDF-15 treatment. Studies were performed four times per condition and repeated in three separate experiments. (D and E) Rate of macrophage apoptosis (D; percentage annexin V + /PI − cells) and necrosis (E; percentage annexin V + /PI + cells) after treatment with 10 ng/ml GDF-15 or 50 µg/ml ox-LDL in both GDF-15 −/− (black bars) and WT (white bars) macrophages. **, P < 0.01; ***, P < 0.001 when compared with control; #, P < 0.05, compared with WT. (F) Phagocytosis capacity in WT (white bars) and GDF-15 −/− (black bars) macrophages. *, P < 0.05 when compared with WT. Bone marrow–derived macrophages from WT and GDF-15 chimeras were pooled and used for apoptosis and phagocytosis assays. Each experiment was performed four times. Error bars are depicted as SEM.

Article Snippet: Serum-deprived RAW 264.7 macrophages were stimulated with 10 ng/ml of recombinant GDF-15 and 100 ng/ml of recombinant mouse TGFβRII/mouse FC (R&D Systems) for 6, 12, and 24 h. Cells were washed twice in PBS and fixed in ice-cold 70% ethanol for 24 h. Cells were then washed twice in HBSS and resuspended in PBS containing 0.1 mM EDTA, 0.1% Triton X-100, 50 µg/ml RNase A, and 50 µg/ml propidium iodide (PI).

Techniques: TUNEL Assay, Staining, Derivative Assay

Journal: The Journal of Experimental Medicine

Article Title: Growth differentiation factor 15 deficiency protects against atherosclerosis by attenuating CCR2-mediated macrophage chemotaxis

doi: 10.1084/jem.20100370

Figure Lengend Snippet: Pro- and antiinflammatory mediators in GDF-15 −/− cells and chimeras. (A–D) Relative mRNA expression of CCR2 (A), MCP-1 (B), IFN-γ (C), and TGF-β (D) in WT (white bars) and GDF-15 −/− (black bars) macrophages. Values are expressed relative to average expression of GAPDH and HPRT reference genes. (E and F) MCP-1 (E) and TGF-β (F) production in WT (white bars) and GDF-15 −/− (black bars) macrophages after LPS treatment. (G) Basal levels of MCP-1 (white bars) and TGF-β (black bars) in WT and GDF-15 −/− chimeras after 4 wk of Western type diet. (H) Relative mRNA expression of PAI-1 and MCP-1 in response to 10 ng/ml GDF-15– and 15 ng/ml TGF-β1–treated WT macrophages. (I) Relative MCP-1 mRNA expression after SMAD-3 inhibition (SIS3; 3 µM) and α-TGFβRI and α-TGFβRII treatment (100 ng/ml) in WT macrophages. Bone marrow–derived macrophages from WT and GDF-15 were pooled and used for RNA expression. Each experiment was done four times. *, P < 0.05; **, P < 0.01. Error bars are depicted as SEM.

Article Snippet: Serum-deprived RAW 264.7 macrophages were stimulated with 10 ng/ml of recombinant GDF-15 and 100 ng/ml of recombinant mouse TGFβRII/mouse FC (R&D Systems) for 6, 12, and 24 h. Cells were washed twice in PBS and fixed in ice-cold 70% ethanol for 24 h. Cells were then washed twice in HBSS and resuspended in PBS containing 0.1 mM EDTA, 0.1% Triton X-100, 50 µg/ml RNase A, and 50 µg/ml propidium iodide (PI).

Techniques: Expressing, Western Blot, Inhibition, Derivative Assay, RNA Expression

Journal: The Journal of Experimental Medicine

Article Title: Growth differentiation factor 15 deficiency protects against atherosclerosis by attenuating CCR2-mediated macrophage chemotaxis

doi: 10.1084/jem.20100370

Figure Lengend Snippet: GDF-15 sensitizes CCR2-mediated chemotactic response. (A) Migration response of WT (white bars) and GDF-15 −/− (black bars) macrophages to GDF-15, MCP-1, and fMLP. (B) Migratory response of WT (white bars) and GDF-15 −/− (black bars) macrophages to GDF-15 after treatment with α-TGFβRI, α-TGFβRII, and SMAD-3 inhibition. (C) Migratory response of WT peritoneal macrophages after combined GDF-15 and MCP-1 treatment. (D) Macrophage migration toward GDF-15 in WT and CCR1-, CCR2-, and CCR5-deficient macrophages. (E) Relative GRK-2 mRNA expression in WT bone marrow–derived macrophages after exposure to GDF-15. (F) Migratory response toward GDF-15 and MCP-1 of GRK-2 +/− macrophages. *, P < 0.05; **, P < 0.01; ***, P < 0.001, when compared with control; and #, P < 0.05; ## , P < 0.01; ### , P < 0.001, when compared with GDF-15. Migration and mRNA expression assays were performed with pooled bone marrow–derived macrophages from WT and GDF-15. Each experiment was repeated six (migration) and four (mRNA) times. Error bars are depicted as SEM.

Article Snippet: Serum-deprived RAW 264.7 macrophages were stimulated with 10 ng/ml of recombinant GDF-15 and 100 ng/ml of recombinant mouse TGFβRII/mouse FC (R&D Systems) for 6, 12, and 24 h. Cells were washed twice in PBS and fixed in ice-cold 70% ethanol for 24 h. Cells were then washed twice in HBSS and resuspended in PBS containing 0.1 mM EDTA, 0.1% Triton X-100, 50 µg/ml RNase A, and 50 µg/ml propidium iodide (PI).

Techniques: Migration, Inhibition, Expressing, Derivative Assay