Journal: Biomedicines

Article Title: THBS1 Induces Dysfunction of Ovarian Granulosa Cells in Patients with Polycystic Ovary Syndrome by Activating the TGF-β/Smad Pathway

doi: 10.3390/biomedicines14061273

Figure Lengend Snippet: THBS1 is up-regulated in the follicular fluid and granulosa cells of PCOS patients and correlates with anti-Müllerian hormone and luteinising hormone. ( A ) Concentrations of THBS1 and IL-6 in follicular fluid from control and PCOS patients were measured by ELISA. individual data points are shown as symbols. ( B ) Relative mRNA expression levels of THBS1 and IL-6 in granulosa cells from control and PCOS patients were determined by qPCR. ( C ) Correlation analyses between follicular fluid THBS1 concentrations and serum AMH and LH levels in PCOS patients. ( D ) Immunofluorescence staining of granulosa cells for FSHR (red) and DAPI (blue). The merged image confirms granulosa cell identity. Scale bar = 50 μm.

Article Snippet: Finally, to determine whether the cellular dysfunction induced by THBS1 overexpression depends on the TGF-β receptor, we treated THBS1-OE KGN cells with the type-I receptor (ALK4/5/7) inhibitor SB-431542 (MedChemExpress HY-10431, 10 μM, 48 h).

Techniques: Control, Enzyme-linked Immunosorbent Assay, Expressing, Immunofluorescence, Staining

Journal: Biomedicines

Article Title: THBS1 Induces Dysfunction of Ovarian Granulosa Cells in Patients with Polycystic Ovary Syndrome by Activating the TGF-β/Smad Pathway

doi: 10.3390/biomedicines14061273

Figure Lengend Snippet: AAV9-mediated knockdown of ovarian Thbs1 ameliorates the PCOS-like reproductive phenotype in a DHEA-induced rat model. ( A ) The timeline schematic was created using FigDraw ( https://www.figdraw.com ), illustrating DHEA-induced PCOS establishment and AAV9-mediated THBS1 knockdown intervention. ( B ) Representative HE staining and immunohistochemistry images of ovarian sections. Scale bar = 500 μm. ( C ) Quantification of time spent in each estrous cycle stage. ( D ) Vaginal smear cytology showing estrous cycle stages. Scale bar = 200 μm. ( E ) Serum hormone levels (AMH, FSH, LH, E2, T) measured by ELISA. Different colored bars represent different experimental groups, different symbols indicate individual animal data point. ( F ) Body weight changes during the 28-day protocol. ( G ) Western blot analysis of ovarian THBS1, BAX, and BCL-2 protein expression. ( H ) AAV9-mediated ovarian THBS1 knockdown (37.4% protein reduction, p = 0.006), band intensity was calculated by Image J.

Article Snippet: Finally, to determine whether the cellular dysfunction induced by THBS1 overexpression depends on the TGF-β receptor, we treated THBS1-OE KGN cells with the type-I receptor (ALK4/5/7) inhibitor SB-431542 (MedChemExpress HY-10431, 10 μM, 48 h).

Techniques: Knockdown, Staining, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Western Blot, Expressing

Journal: Biomedicines

Article Title: THBS1 Induces Dysfunction of Ovarian Granulosa Cells in Patients with Polycystic Ovary Syndrome by Activating the TGF-β/Smad Pathway

doi: 10.3390/biomedicines14061273

Figure Lengend Snippet: THBS1 overexpression impairs KGN Cells function. ( A , B ) Validation of THBS1 overexpression by Western blot and qPCR. ( C ) CCK-8 assay showing suppressed cell proliferation over 3 days. ( D , E ) Flow cytometry analysis of apoptosis. ( F , G ) Flow cytometry analysis of cell cycle distribution. ( H – J ) Western blot analysis of apoptosis markers (BAX, BCL-2), inflammatory cytokines (IL-6, TNF-α), steroidogenic enzymes (CYP19, CYP17).

Article Snippet: Finally, to determine whether the cellular dysfunction induced by THBS1 overexpression depends on the TGF-β receptor, we treated THBS1-OE KGN cells with the type-I receptor (ALK4/5/7) inhibitor SB-431542 (MedChemExpress HY-10431, 10 μM, 48 h).

Techniques: Over Expression, Biomarker Discovery, Western Blot, CCK-8 Assay, Flow Cytometry

Journal: Biomedicines

Article Title: THBS1 Induces Dysfunction of Ovarian Granulosa Cells in Patients with Polycystic Ovary Syndrome by Activating the TGF-β/Smad Pathway

doi: 10.3390/biomedicines14061273

Figure Lengend Snippet: Molecular docking and 100-ns dynamics simulation reveal a stable association between THBS1 and human latent TGF-1 (PDB 9VJJ). ( A ) Surface representation of THBS1-latent TGF-β1 (PDB 9VJJ) binding interface. Brownish-yellow: TGF-β1, purple-blue: THBS1, red: interfacial binding residues. ( B ) Docking scores and confidence scores of top models. ( C ) Detailed interaction analysis showing hydrogen bonds (blue lines) and salt bridges (yellow dashed lines) between THBS1 (blue) and latent TGF-β1 (PDB 9VJJ) (yellow). ( D ) Per-residue MM/PBSA decomposition. ( E ) RMSD analysis of the protein complex over 100 ns. ( F ) Radius of gyration (Rg) analysis showing structural compactness. ( G ) Solvent-accessible surface area (SASA) analysis. ( H ) Hydrogen bond count throughout the simulation. RMSF analysis of THBS1 ( I ) and latent TGF-β1 (PDB 9VJJ) ( J ) residues. ( K , L ) Gibbs free-energy landscape (FEL) based on RMSD and Rg. ( M ) MM/PBSA binding free energy decomposition.

Article Snippet: Finally, to determine whether the cellular dysfunction induced by THBS1 overexpression depends on the TGF-β receptor, we treated THBS1-OE KGN cells with the type-I receptor (ALK4/5/7) inhibitor SB-431542 (MedChemExpress HY-10431, 10 μM, 48 h).

Techniques: Binding Assay, Residue, Solvent

Journal: Biomedicines

Article Title: THBS1 Induces Dysfunction of Ovarian Granulosa Cells in Patients with Polycystic Ovary Syndrome by Activating the TGF-β/Smad Pathway

doi: 10.3390/biomedicines14061273

Figure Lengend Snippet: THBS1 overexpression activates the TGF-β1/Smad axis. ( A ) DESeq2 volcano (267 up + 317 down at |log FC| > 1, Padj < 0.05). ( B ) Hierarchical clustering of differentially expressed genes. ( C ) TGF-β/Smad pathway gene expression (28 canonical genes). *, **, *** Data are presented as mean ± SEM. ( D ) Reciprocal co-immunoprecipitation: lanes IgG/IP/Input. ( E ) Active TGF-β1—patient FF. Different colored bars represent different experimental groups, and different symbols indicate individual sample data points. ( F ) Active/Total ratio--patient FF. ( G ) Active TGF-β1—rat serum. ( H ) Active/Total ratio—rat serum. ( I ) Western blot analysis of TGF-β/Smad—KGN cells (THBS1-OE). ( J , K ) Flow cytometry apoptosis (THBS1-OE ± SB-431542). ( L – N ) Western blot analysis of apoptosis markers (BAX, BCL-2), inflammatory cytokines (IL-6, TNF-α), steroidogenic enzymes (CYP19, CYP17), and TGF-β/SMAD2 signaling pathway components.

Article Snippet: Finally, to determine whether the cellular dysfunction induced by THBS1 overexpression depends on the TGF-β receptor, we treated THBS1-OE KGN cells with the type-I receptor (ALK4/5/7) inhibitor SB-431542 (MedChemExpress HY-10431, 10 μM, 48 h).

Techniques: Over Expression, Gene Expression, Immunoprecipitation, Western Blot, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: Integrated transcriptomic profiling of programmed cell death patterns unveils macrophage-hepatocyte crosstalk via THBS1-CD47 axis in hepatic ischemia-reperfusion injury

doi: 10.3389/fimmu.2026.1769849

Figure Lengend Snippet: OGD/R model induced THBS1 expression in RAW264.7 cells and apoptosis in AML12 cells. (A) Schematic of OGD/R treatment. AML12 cells (lower chamber) and RAW264.7 cells (upper chamber) were co-cultured in serum-free, glucose-free DMEM under hypoxic conditions (1% O2) for 6 hours, followed by re-oxygenation in complete medium for another 3 hours. Control cells were cultured in complete medium under normoxic conditions. (B) THBS1 protein expression in RAW264.7 cells between Control and OGD/R groups. (C) The contents of IL-1β, IL-6, and TNF-α in RAW264.7 cell culture medium between Control and OGD/R groups. (D) The expressions of pro-apoptotic proteins Bax, Cleaved-caspase 3, and anti-apoptotic Bcl2 protein in AML12 cells between Control and OGD/R groups. (E) Cell death in the Control and OGD/R groups was assessed using Hoechst 33342 and PI double staining. Scale bar = 50μm. (F) The mitochondrial membrane potential (ΔψM) of the Control and OGD/R groups was detected using JC-1 staining. Scale bar = 50μm.

Article Snippet: Furthermore, AML12 cells were seeded in 6-well plates 24 hours prior to the establishment of the OGD/R model, with each well receiving 1 μg/mL of recombinant mouse THBS1 protein (MCE, Shanghai, China).

Techniques: Expressing, Cell Culture, Control, Double Staining, Membrane, Staining

Journal: Frontiers in Immunology

Article Title: Integrated transcriptomic profiling of programmed cell death patterns unveils macrophage-hepatocyte crosstalk via THBS1-CD47 axis in hepatic ischemia-reperfusion injury

doi: 10.3389/fimmu.2026.1769849

Figure Lengend Snippet: RAW264.7-specific THBS1 siRNA treatment alleviated OGD/R-induced apoptosis in AML12 cells. (A) Schematic of OGD/R treatment. RAW264.7 cells, separately transfected with siRNA-NC and siRNA-THBS1, were seeded in the upper chamber and co-cultured with AML12 cells (lower chamber) under hypoxic conditions (1% O2) in serum-free, glucose-free DMEM for 6 hours, followed by reoxygenation in complete medium for another 3 hours. (B) THBS1 protein expression in RAW264.7 cells between siRNA-NC and siRNA-THBS1 groups. (C) The contents of IL-1β, IL-6, and TNF-α in RAW264.7 cell culture medium between siRNA-NC and siRNA-THBS1 groups. (D) The expression of pro-apoptotic proteins Bax, Cleaved-caspase 3, and anti-apoptotic Bcl2 protein in AML12 cells between siRNA-NC and siRNA-THBS1 groups. (E) Cell death in the siRNA-NC and siRNA-THBS1 groups was assessed using Hoechst 33342 and PI double staining. Scale bar = 50μm. (F) The mitochondrial membrane potential (ΔψM) of the siRNA-NC and siRNA-THBS1 groups was detected using JC-1 staining. Scale bar = 50μm.

Article Snippet: Furthermore, AML12 cells were seeded in 6-well plates 24 hours prior to the establishment of the OGD/R model, with each well receiving 1 μg/mL of recombinant mouse THBS1 protein (MCE, Shanghai, China).

Techniques: Transfection, Cell Culture, Expressing, Double Staining, Membrane, Staining

Journal: Frontiers in Immunology

Article Title: Integrated transcriptomic profiling of programmed cell death patterns unveils macrophage-hepatocyte crosstalk via THBS1-CD47 axis in hepatic ischemia-reperfusion injury

doi: 10.3389/fimmu.2026.1769849

Figure Lengend Snippet: THBS1 promoted hepatocyte apoptosis by inhibiting PI3K-AKT- NF-κB signaling. (A) The expression of p-AKT, AKT, p-NF-κB p65 and NF-κB p65 proteins in AML12 cells between siRNA-NC and siRNA-THBS1 groups. (B) The expression of pro-apoptotic proteins Bax, Cleaved-caspase 3, and anti-apoptotic Bcl2 protein in AML12 cells among the siRNA-NC, siRNA-THBS1 and siRNA-THBS1+ LY294002 groups. (C) Cell death in the siRNA-NC, siRNA-THBS1 and siRNA-THBS1+ LY294002 groups was assessed using Hoechst 33342 and PI double staining. Scale bar = 50μm. (D) The mitochondrial membrane potential (ΔψM) of the siRNA-NC, siRNA-THBS1 and siRNA-THBS1+ LY294002 was detected using JC-1 staining. Scale bar = 50μm.

Article Snippet: Furthermore, AML12 cells were seeded in 6-well plates 24 hours prior to the establishment of the OGD/R model, with each well receiving 1 μg/mL of recombinant mouse THBS1 protein (MCE, Shanghai, China).

Techniques: Expressing, Double Staining, Membrane, Staining

Journal: Frontiers in Immunology

Article Title: Integrated transcriptomic profiling of programmed cell death patterns unveils macrophage-hepatocyte crosstalk via THBS1-CD47 axis in hepatic ischemia-reperfusion injury

doi: 10.3389/fimmu.2026.1769849

Figure Lengend Snippet: THBS1-mediated hepatocyte apoptosis depended on the CD47 receptor. (A) CD47 protein expression in AML12 cells between Control and OGD/R groups. (B) Schematic of OGD/R treatment. AML12 cells, separately transfected with siRNA-NC and siRNA-CD47, were seeded in the lower chamber and co-cultured with RAW264.7 cells (upper chamber) under hypoxic conditions (1% O2) in serum-free, glucose-free DMEM for 6 hours, followed by reoxygenation in complete medium for another 3 hours. (C) The expression of pro-apoptotic proteins Bax, Cleaved-caspase 3, and anti-apoptotic Bcl2 protein in AML12 cells between siRNA-NC and siRNA-CD47 groups. (D) Cell death in the siRNA-NC and siRNA-CD47 groups was assessed using Hoechst 33342 and PI double staining. Scale bar = 50μm. (E) Schematic of OGD/R treatment. AML12 cells were separately transfected with siRNA-NC and siRNA-CD47, respectively, then cultured in serum-free, glucose-free DMEM supplemented with 1 ng/mL rmTHBS1 under hypoxic conditions (1% O2) for 6 hours, followed by reoxygenation in complete medium for another 3 hours. (F) The expression of pro-apoptotic proteins Bax, Cleaved-caspase 3, and anti-apoptotic Bcl2 protein in AML12 cells among the siRNA-NC, siRNA-NC+ rmTHBS1 and siRNA-CD47+ rmTHBS1 groups. (G) Cell death in the siRNA-NC, siRNA-NC+ rmTHBS1 and siRNA-CD47+ rmTHBS1 groups was assessed using Hoechst 33342 and PI double staining. Scale bar = 50μm. (H) Schematic diagram of the THBS1-CD47 axis’s role in macrophage-hepatocyte crosstalk during HIRI.

Article Snippet: Furthermore, AML12 cells were seeded in 6-well plates 24 hours prior to the establishment of the OGD/R model, with each well receiving 1 μg/mL of recombinant mouse THBS1 protein (MCE, Shanghai, China).

Techniques: Expressing, Control, Transfection, Cell Culture, Double Staining