Journal: Investigative Ophthalmology & Visual Science

Article Title: Corneal Nerves Promote Alkali Burn Repair by Modulating Macrophages and Neutrophils via Calcitonin Gene-Related Peptide

doi: 10.1167/iovs.67.4.60

Figure Lengend Snippet: CGRP modulates macrophage function via the cAMP/TSP-1 signaling pathway ( n = 4). (A) Schematic of the cAMP signaling pathway downstream of the CGRP-CALCRL/RAMP1 axis. Activation of adenylyl cyclase (AC) elevates the intracellular level of cAMP. (B) The concentration of cAMP in cell lysates was measured by a competitive ELISA after in vitro stimulation of macrophages with CGRP or co-stimulation of CGRP with SQ22536 for 30 minutes ( n = 4). (C) Bar graph showing quantification of apoptosis in macrophages following in vitro treatment with CGRP alone or in combination with SQ22536 ( n = 4). (D, E) The expression of CD206 and Arg-1 in macrophages after treatment with CGRP or CGRP combined with SQ22536 ( n = 4). (F) Macrophage efferocytosis after CGRP or CGRP + SQ22536 treatment ( n = 4). (G) Volcano plot showing upregulation of thbs1 gene expression in CGRP-treated macrophages. (H, J) Representative micrographs and quantification of increased TSP-1 expression in macrophages following CGRP or CGRP + SQ22536 stimulation in vitro ( n = 4). (I, K) Representative micrographs and quantification of TSP-1+ macrophages in the cornea following 7 days of topical CGRP treatment ( n = 4). (L) Quantification of apoptosis in macrophages treated with TSP-1 in vitro ( n = 4). (M, N) The expression of CD206 and Arg-1 in macrophages after treatment with TSP-1 ( n = 4). (O) Macrophage efferocytosis after CGRP or CGRP + SQ22536 treatment. Data are shown as mean ± SD. ns, not significant; * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: For TSP-1-induced apoptosis, BMDMs were incubated with RPMI-1640 medium containing 100 nM TSP-1 (HY-P701325; MedChenExpress) for 24 hours.

Techniques: Activation Assay, Concentration Assay, Competitive ELISA, In Vitro, Expressing, Gene Expression

Journal: Oncology Reports

Article Title: Combination of ivermectin and metformin promotes autophagy in MCF-7 cells by inhibiting phosphorylation of the PI3K/AKT/mTOR pathway

doi: 10.3892/or.2026.9136

Figure Lengend Snippet: Western blotting detection of THBS1 protein expression in MCF-7 cells. (A) Representative western blotting image shows the expression of THBS1 protein in MCF-7 cells was downregulated. The molecular weights of THBS1 and β-actin are indicated as 180 and 42 kDa, respectively. (B) Semi-quantification of the western blotting results. IVM, ivermectin (6 µM); MET, metformin (6 mM); MCF-7, human mammary tumor cell line; Ctrl, control; +, with treatment. All data were measured as mean ± SEM by three independent experiments. **P<0.01.

Article Snippet: The present study identified the CDS region of THBS1 from the National Centre for Biotechnology Information (NCBI) database.

Techniques: Western Blot, Expressing, Control

Journal: Oncology Reports

Article Title: Combination of ivermectin and metformin promotes autophagy in MCF-7 cells by inhibiting phosphorylation of the PI3K/AKT/mTOR pathway

doi: 10.3892/or.2026.9136

Figure Lengend Snippet: Overexpression of THBS1 enhances the phosphorylation level of proteins in the PI3K/AKT/mTOR pathway. (A) Schematic diagram of THBS1 overexpression plasmid construction. (B) Detection of THBS1 CDS region amplification by agarose gel electrophoresis. (C) Representative western blotting images and (D) semi-quantification of THBS1 overexpression efficiency in MCF-7 cells. MCF-7, human mammary tumor cell line. All data were measured as mean ± SEM by three independent experiments. **P<0.01.

Article Snippet: The present study identified the CDS region of THBS1 from the National Centre for Biotechnology Information (NCBI) database.

Techniques: Over Expression, Phospho-proteomics, Plasmid Preparation, Amplification, Agarose Gel Electrophoresis, Western Blot

Journal: Cell Death & Disease

Article Title: Vascular SMC-like CAF-derived THBS1 drives tumor-associated neutrophil recruitment to orchestrate an immunosuppressive microenvironment in gastric cancer

doi: 10.1038/s41419-026-08945-1

Figure Lengend Snippet: A UMAP plots of major cell types from tumor and non-malignant normal tissues of GC patients in the GSE183904 dataset. B Expression of canonical marker genes used to annotate major cell lineages. C UMAP visualization of 11,353 fibroblasts from the GSE183904 dataset, clustered into six distinct subtypes. D Heatmap showing the top differentially upregulated genes in each fibroblast subcluster. E UMAP plots depicting the expression of canonical marker genes across fibroblast subclusters. F UMAP analysis of fibroblast subclusters derived from tumor versus non-malignant normal samples in GSE183904 . G Proportional composition of each fibroblast subcluster in tumor and normal tissues. H UMAP plot showing THBS1 expression across fibroblast subtypes in GSE183904 . I THBS1 was significantly upregulated in the Fibro_1 subtype. ***, P < 0.001.

Article Snippet: The antibodies and corresponding TSA colors used were: E-cadherin (GB12083, yellow, Servicebio), THBS1 (#2125, green, ABclonal), MMP9 (GB11132-100, cyan, Servicebio), and COL1A1 (GB11022, red, Servicebio).

Techniques: Expressing, Marker, Derivative Assay

Journal: Cell Death & Disease

Article Title: Vascular SMC-like CAF-derived THBS1 drives tumor-associated neutrophil recruitment to orchestrate an immunosuppressive microenvironment in gastric cancer

doi: 10.1038/s41419-026-08945-1

Figure Lengend Snippet: A UMAP plot of single cells from gastric tissues annotated using the DISCO database. B, C THBS1 is mainly expressed in monocytes, smooth muscle cells (SMC), fibroblasts, and gastric epithelial glandular chief cells. D IHC results showed that THBS1 was mainly expressed in chief cells, SMC and VSMC cells. E Representative IHC staining of THBS1 in normal gastric tissues and GC tissues. THBS1 is mainly expressed in chief cells in normal gastric tissues, but is widely expressed in nearly all epithelial cells in GC. F IHC assays confirmed that THBS1 was overexpressed in GC ( n = 12). G–I THBS1 mRNA was overexpressed in the GSE179252 ( n = 38), GSE184336 ( n = 230), and GSE122401 ( n = 80) stomach cancer cohorts. **, p < 0.01, ***, p < 0.001.

Article Snippet: The antibodies and corresponding TSA colors used were: E-cadherin (GB12083, yellow, Servicebio), THBS1 (#2125, green, ABclonal), MMP9 (GB11132-100, cyan, Servicebio), and COL1A1 (GB11022, red, Servicebio).

Techniques: Immunohistochemistry

Journal: Cell Death & Disease

Article Title: Vascular SMC-like CAF-derived THBS1 drives tumor-associated neutrophil recruitment to orchestrate an immunosuppressive microenvironment in gastric cancer

doi: 10.1038/s41419-026-08945-1

Figure Lengend Snippet: A Genes regulated by SNAI1/2 (log2FC > 1) were identified based on RNA-seq data ( GSE161551 ) from GC cells with or without SNAI1/2 overexpression. B Genes regulated by SNAI1 or SNAI2 were clustered into five subclusters. SNAI2 has a stronger transcriptional regulatory effect than SNAI1 on genes in clusters C3-C5. Cluster C1 and C4 represent genes significantly regulated by SNAI2 alone. C Promoter analysis revcealed multiple SNAI2 binding motifs in the THBS1 promoter. D Chip-seq analysis of SNAI2 using the Cistrome web tool showed significant binding of SNAI2 to the THBS1 promoter. E The EMT subtype of GC in the GSE62254 cohort exhibited the highest THBS1 expression. F THBS1 expression was positively correlated with EMT signaling in the TCGA_STAD cohort. G THBS1 was highly co-expressed with SNAI2 in both the TCGA_STAD and GSE62254 cohorts. H, I CHIP-seq and qPCR analyses showed that SNAI2 directly binds to the THBS1 promoter in GC cells. J–L SNAI2 mRNA was overexpressed in the GSE179252 ( n = 38), GSE184336 ( n = 230), and GSE122401 ( n = 80) stomach cancer cohorts. M SNAI2 overexpression predicted poor prognosis in the TCGA_STAD cohort. ***, p < 0.001, ****, p < 0.0001.

Article Snippet: The antibodies and corresponding TSA colors used were: E-cadherin (GB12083, yellow, Servicebio), THBS1 (#2125, green, ABclonal), MMP9 (GB11132-100, cyan, Servicebio), and COL1A1 (GB11022, red, Servicebio).

Techniques: RNA Sequencing, Over Expression, Binding Assay, ChIP-sequencing, Expressing

Journal: Cell Death & Disease

Article Title: Vascular SMC-like CAF-derived THBS1 drives tumor-associated neutrophil recruitment to orchestrate an immunosuppressive microenvironment in gastric cancer

doi: 10.1038/s41419-026-08945-1

Figure Lengend Snippet: A THBS1 expression was higher in diffuse-type GC patients than in intestinal GC patients in the TCGA_STAD cohort. B The expression of THBS1 is positively correlated with poor differentiation in the TCGA_STAD cohort. C, D THBS1 expression is highly expressed in advanced-stage GC and lowly expressed in early-stage GC in the TCGA_STAD cohort. E In the GSE62254 cohort, THBS1 was highly expressed in diffuse GC. F–I The expression level of THBS1 was positively correlated with pathological stage, T stage, lymph node metastasis, and perineural invasion in the GSE62254 GC patients. J THBS1 overexpression predicted poor overall survival (OVS) and disease-free survival (DFS) in the TCGA_STAD cohort. K GC patients with high THBS1 expression had shorter OVS and DFS in the GSE62254 cohort. *, p < 0.05, **, p < 0.01. ***, p < 0.001, ****, p < 0.0001.

Article Snippet: The antibodies and corresponding TSA colors used were: E-cadherin (GB12083, yellow, Servicebio), THBS1 (#2125, green, ABclonal), MMP9 (GB11132-100, cyan, Servicebio), and COL1A1 (GB11022, red, Servicebio).

Techniques: Expressing, Over Expression

Journal: Cell Death & Disease

Article Title: Vascular SMC-like CAF-derived THBS1 drives tumor-associated neutrophil recruitment to orchestrate an immunosuppressive microenvironment in gastric cancer

doi: 10.1038/s41419-026-08945-1

Figure Lengend Snippet: A To evaluate changes in the immune microenvironment during GC progression, we performed deconvolution analysis using the ssGSEA algorithm on transcriptomic data from the GSE122401 cohort, which included tumor and paired adjacent tissues from 80 patients. B Heatmap showing infiltration scores of 24 immune cell types in GC and adjacent normal tissues. C A total of 19 immune cell types, including neutrophils, showed significant changes in infiltration during GC progression. D MMP9 is a marker gene for neutrophils. IHC staining of GC and adjacent normal tissues using an anti-MMP9 antibody revealed neutrophil. The blue arrows represent neutrophils. E Neutrophils infiltration was significantly increased in GC. F THBS1 expression (log 2 ) was positively correlated with neutrophils infiltration in the GSE122401 cohort. G Deconvolution analysis using the ssGSEA algorithm on the TCGA_STAD transcriptome revealed a significant positive correlation between THBS1 expression and neutrophil infiltration. H–J Prognostic analysis of 24 immune cell types showed that GC patients with high infiltration of macrophages and neutrophils had poor prognosis in the TCGA_STAD cohort. K In the TCGA_SATD cohort, the expression level of THBS1 was significantly co-expressed with neutrophil surface marker genes and secreted protein genes. **, p < 0.01. ***, p < 0.001.

Article Snippet: The antibodies and corresponding TSA colors used were: E-cadherin (GB12083, yellow, Servicebio), THBS1 (#2125, green, ABclonal), MMP9 (GB11132-100, cyan, Servicebio), and COL1A1 (GB11022, red, Servicebio).

Techniques: Marker, Immunohistochemistry, Expressing

Journal: Cell Death & Disease

Article Title: Vascular SMC-like CAF-derived THBS1 drives tumor-associated neutrophil recruitment to orchestrate an immunosuppressive microenvironment in gastric cancer

doi: 10.1038/s41419-026-08945-1

Figure Lengend Snippet: A, B IHC and RNA-seq analysis confirmed successful THBS1 knockdown in CAFs. C Schematic diagram of co-culture system for CAFs and GC Cells. D–G Knockdown of THBS1 in CAFs significantly attenuated their ability to promote GC cell proliferation in vitro. H–J In a xenograft tumor model, CAF cells and GC cells were co-inoculated subcutaneously into nude mice at a 1:1 ratio. CAFs with THBS1 knockdown significantly suppressed xenograft tumor size, weight, and volume. K No significant difference in body weight was observed between the control and the THBS1-knockdown groups. L, M Knockdown of THBS1 in CAFs significantly reduced neutrophil infiltration in xenograft tumors. **, p < 0.01, ***, p < 0.001.

Article Snippet: The antibodies and corresponding TSA colors used were: E-cadherin (GB12083, yellow, Servicebio), THBS1 (#2125, green, ABclonal), MMP9 (GB11132-100, cyan, Servicebio), and COL1A1 (GB11022, red, Servicebio).

Techniques: RNA Sequencing, Knockdown, Co-Culture Assay, In Vitro, Control

Journal: Cell Death & Disease

Article Title: Vascular SMC-like CAF-derived THBS1 drives tumor-associated neutrophil recruitment to orchestrate an immunosuppressive microenvironment in gastric cancer

doi: 10.1038/s41419-026-08945-1

Figure Lengend Snippet: A, B qPCR and western blotting assays confirmed successful THBS1 overexpression in the AGS GC cell line. C–E CCK-8, colony formation, and EdU staining assays showed that THBS1 overexpression promotes AGS cell proliferation. F, G Transwell invasion and wound healing assays demonstrated that THBS1 overexpression enhanced AGS cell invasion and migration. H–J Equal amounts of control and THBS1-overexpressing AGS cells were subcutaneously inoculated into nude mice. THBS1 overexpression significantly increased xenograft tumor size, weight, and volume. K No significant difference in body weight was observed between the control and the THBS1 overexpression groups. L, M Neutrophil infiltration was significantly higher in xenograft tumors from the THBS1-overexpression group than in the control group. **, p < 0.01.

Article Snippet: The antibodies and corresponding TSA colors used were: E-cadherin (GB12083, yellow, Servicebio), THBS1 (#2125, green, ABclonal), MMP9 (GB11132-100, cyan, Servicebio), and COL1A1 (GB11022, red, Servicebio).

Techniques: Western Blot, Over Expression, CCK-8 Assay, Staining, Migration, Control

Journal: Cell Death & Disease

Article Title: Vascular SMC-like CAF-derived THBS1 drives tumor-associated neutrophil recruitment to orchestrate an immunosuppressive microenvironment in gastric cancer

doi: 10.1038/s41419-026-08945-1

Figure Lengend Snippet: A, B In the TCGA_STAD cohort, GC subtypes with high CAF infiltration showed elevated neutrophil infiltration and THBS1 expression. C We established a GC patient-derived PDX model in nude mice and randomly assigned mice to receive either THBS1 monoclonal antibody or control treatment. mIHC was performed on PDX xenografts to assess the effect of THBS1 blockade on neutrophil infiltration. mIHC results revealed significant co-localization of neutrophils and fibroblasts. D Neutrophil infiltration was significantly reduced in xenografts from the THBS1 monoclonal antibody–treated group. E Tumor volume measurements in the PDX model comparing the THBS1 monoclonal antibody–treated and control groups. F RNA sequencing of THBS1-knockdown and control CAF cells revealed differentially expressed genes, with multiple chemokines highlighted in the heatmap. G Genes significantly downregulated upon THBS1 knockdown were enriched in pathways related to neutrophil chemotaxis and migration. H Gene expression analysis showed that among the chemokines regulated by THBS1, the expression levels of CXCL1, CXCL5, and CXCL6 were significantly upregulated in GC. I–K ELISA assays on CAF culture supernatants confirmed elevated secretion of CXCL1, CXCL5, and CXCL6. **, p < 0.01, ***, p < 0.001.

Article Snippet: The antibodies and corresponding TSA colors used were: E-cadherin (GB12083, yellow, Servicebio), THBS1 (#2125, green, ABclonal), MMP9 (GB11132-100, cyan, Servicebio), and COL1A1 (GB11022, red, Servicebio).

Techniques: Expressing, Derivative Assay, Control, RNA Sequencing, Knockdown, Chemotaxis Assay, Migration, Gene Expression, Enzyme-linked Immunosorbent Assay

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