Journal: Research

Article Title: Mitophagy Reprograms Lactate Metabolism to Suppress THBS1 via H3K18la Reduction, Alleviating Intervertebral Disc Degeneration

doi: 10.34133/research.0957

Figure Lengend Snippet: Down-regulation of lactate production and histone lactylation is driven by mitophagy activation. (A) Lactate levels of the young and aged NP cells treated with or without TJ0113 (5 μM, 48 h; n = 5) after knocking down MCL1. (B) Western blot analysis of PAN-KLA in young and aged NP cells treated with or without TJ0113 (5 μM, 48 h) after knocking down MCL1. (C and D) Western blot analysis of lysine lactylation markers (H3K9la, H3K14la, H3K18la, H4K12la) treated with TJ0113 or not (5 μM, 48 h). Histone H3/H4 served as loading controls. (E) Heatmap depicting H3K18la genomic occupancy profiles [±3 kb flanking transcription start sites (TSSs)] treated with TJ0113 or not (5 μM, 48 h). Genes are ranked vertically by descending H3K18la signal intensity. (F) Comparative pie charts showing genome-wide differential H3K18la distribution patterns at annotated genomic regions in aged NP cells treated with TJ0113 (5 μM, 48 h) compared to untreated controls. (G) KEGG enrichment analysis of H3K18la-marked genes. (H) Venn diagram depicting genes down-regulated in RNA-seq analysis of aged NP cells treated with TJ0113 (5 μM, 48 h) compared to untreated controls and the down-regulated target genes bound by H3K18la under identical treatment conditions. (I) Normalized read densities for H3K18la at the THBS1 genes. (J) ChIP-qPCR assay in aged NP cells. Results represent the mean ± SD of at least 3 independent experiments. Significance levels are shown within the graphs.

Article Snippet: FX-11 (catalog no. HY-16214), L-Lac (catalog no. HY-Y0479), and THBS1 recombinant protein (catalog no. HY- P70725 ) were purchased from MedChemExpress.

Techniques: Activation Assay, Western Blot, Genome Wide, RNA Sequencing, ChIP-qPCR

Journal: Research

Article Title: Mitophagy Reprograms Lactate Metabolism to Suppress THBS1 via H3K18la Reduction, Alleviating Intervertebral Disc Degeneration

doi: 10.34133/research.0957

Figure Lengend Snippet: The mitophagy-driven metabolic–epigenetic axis suppresses SASP factors in senescent NP cells. (A) Western blot analysis of SASP factors and thrombospondin-1 (THBS1) in young and aged NP cells. (B) Schematic illustration of the experimental design. (C) Western blot analysis of SASP factors and THBS1 in aged NP cells treated with vehicle, TJ0113 (5 μM), FX-11(20 μM), or both. (D) Western blot analysis of SASP factors and THBS1 in aged NP cells treated with vehicle, TJ0113 (5 μM), L-Lac (10 μM), or both. (E) qRT-PCR analysis of SASP factors in aged NP cells with treatment as (B) and (C) ( n = 3). (F) Western blot analysis of SASP factors and THBS1 in control and THBS1 knockdown aged NP cells treated with or without TJ0113 (5 μM, 48 h). (G) qRT-PCR analysis of SASP factors in control and THBS1 knockdown aged NP cells treated with TJ0113 or not (5 μM, 48 h) ( n = 3). (H) Western blot analysis of SASP factors and THBS1 in control and THBS1-treated (0.1/1 μg/ml, 48 h) aged NP cells under treatment with or without TJ0113 (5 μM, 48 h). (I) qRT-PCR analysis of SASP factors in control and THBS1-treated (0.1/1 μg/ml, 48 h) aged NP cells under treatment with or without TJ0113 (5 μM, 48 h) ( n = 3). Results represent the mean ± SD of at least 3 independent experiments. Significance levels are shown within the graphs.

Article Snippet: FX-11 (catalog no. HY-16214), L-Lac (catalog no. HY-Y0479), and THBS1 recombinant protein (catalog no. HY- P70725 ) were purchased from MedChemExpress.

Techniques: Western Blot, Quantitative RT-PCR, Control, Knockdown

Journal: Research

Article Title: Mitophagy Reprograms Lactate Metabolism to Suppress THBS1 via H3K18la Reduction, Alleviating Intervertebral Disc Degeneration

doi: 10.34133/research.0957

Figure Lengend Snippet: Targeting the lactate–H3K18la–THBS1 axis attenuates IVDD. (A) Schematic illustration of the experimental design. (B) Representative images of H&E and SO&FG staining of rat coccygeal IVDs at 12 weeks after treatment. Scale bar, 1 mm. (C) histological score ( n = 6) of rat coccygeal IVDs. (D) Representative immunohistochemistry images of H3K18la in rat coccygeal IVDs at 12 weeks after treatment. Scale bar, 200 μm. (E) Representative IF images of THBS1 in rat coccygeal IVDs at 12 weeks after treatment. Scale bar, 500 μm. (F and G) Quantification of SASP factors in rat coccygeal IVDs. Results represent the mean ± SD of at least 3 independent experiments. Significance levels are shown within the graphs.

Article Snippet: FX-11 (catalog no. HY-16214), L-Lac (catalog no. HY-Y0479), and THBS1 recombinant protein (catalog no. HY- P70725 ) were purchased from MedChemExpress.

Techniques: Staining, Immunohistochemistry

Journal: Journal of ovarian research

Article Title: Thrombospondin-1 induces CD8 + T cell exhaustion and immune suppression within the tumor microenvironment of ovarian cancer.

doi: 10.1186/s13048-025-01668-5

Figure Lengend Snippet: Fig. 1 THBS1 is significantly overexpressed in OC and correlates with poor prognosis. (A) Comparison of THBS1 expression levels between OC tissues (TCGA-OV dataset) and normal ovarian tissues (GTEx dataset). (B) THBS1 expression levels in normal ovarian epithelial cells versus OC cell lines (GSE14407 dataset). (C) Association between THBS1 expression levels and poor prognosis in OC patients (TCGA-OV dataset). (D) IHC analysis of THBS1 staining intensity in tumor and adjacent normal tissues from 97 OC patients. (E–G) Correlation of THBS1 staining intensity with clinical stage, tumor differentia tion, and lymph node metastasis in 97 OC patients. (H) qPCR analysis of THBS1 mRNA expression levels in the ovarian epithelial cell line IOSE and OC cell lines SKOV3, A2780, and OVCAR3. Data are presented as violin plots, with each point representing an experimental replicate or patient sample. Statistical significance was determined using Student’s t-test or one-way ANOVA, followed by Tukey’s multiple comparison test (P < 0.05)

Article Snippet: Recombinant THBS1 protein (R&D Systems) was added to the culture at varying concentrations (0–100 ng/mL).

Techniques: Comparison, Expressing, Staining

Journal: Journal of ovarian research

Article Title: Thrombospondin-1 induces CD8 + T cell exhaustion and immune suppression within the tumor microenvironment of ovarian cancer.

doi: 10.1186/s13048-025-01668-5

Figure Lengend Snippet: Fig. 2 High THBS1 expression positively correlates with tumor-associated macrophage infiltration. (A) Correlation between THBS1 expression and infiltra tion of CD8 + T cells, dendritic cells (DCs), and macrophages, analyzed using TIMER 2.0. (B) IHC analysis of CD68 (a macrophage marker) staining intensity in tumor and adjacent normal tissues from 97 OC patients. (C) Spearman correlation analysis between THBS1 and CD68 staining intensities. (D–E) IHC analysis of PD-L1 and GAL-3 staining intensities in tumor tissues from 97 OC patients. (F) Pearson correlation analysis of THBS1 with PD-L1, GAL-3, and CD68 expression levels in the TCGA-OV dataset. Data are presented as violin plots, with each point representing an experimental replicate or patient sample. Statistical significance was determined using Student’s t-test or one-way ANOVA, followed by Tukey’s multiple comparison test (P < 0.05)

Article Snippet: Recombinant THBS1 protein (R&D Systems) was added to the culture at varying concentrations (0–100 ng/mL).

Techniques: Expressing, Marker, Staining, Comparison

Journal: Journal of ovarian research

Article Title: Thrombospondin-1 induces CD8 + T cell exhaustion and immune suppression within the tumor microenvironment of ovarian cancer.

doi: 10.1186/s13048-025-01668-5

Figure Lengend Snippet: Fig. 3 High THBS1 expression enhances the chemotactic recruitment of M2 macrophages. (A) qPCR analysis of THBS1 mRNA expression after overexpres sion in SKOV3 and A2780 cells. (B) Schematic representation of co-culture experiments between OC cells and PMA-treated THP-1 macrophages. (C) Flow cytometry analysis of F4/80 + CD206 + macrophage populations after co-culture. (D) Immunofluorescence analysis of CD206 intensity in macrophages post-co-culture. (E) ELISA quantification of M2 cytokines (TGF-β1, CCL1, IL-4) and M1 cytokines (TNF-α, IL-1β, IL-6) in culture supernatants. (F) Crystal violet staining to assess chemotactic recruitment of macrophages by OC cells. (G) qPCR analysis of PD-L1 and GAL-3 mRNA levels in SKOV3 and A2780 cells post-co-culture. Data are presented as violin plots, with each point representing an experimental replicate. Statistical significance was determined using two-way ANOVA, followed by Tukey’s multiple comparison test (P < 0.05)

Article Snippet: Recombinant THBS1 protein (R&D Systems) was added to the culture at varying concentrations (0–100 ng/mL).

Techniques: Expressing, Co-Culture Assay, Flow Cytometry, Immunofluorescence, Enzyme-linked Immunosorbent Assay, Staining, Comparison

Journal: Journal of ovarian research

Article Title: Thrombospondin-1 induces CD8 + T cell exhaustion and immune suppression within the tumor microenvironment of ovarian cancer.

doi: 10.1186/s13048-025-01668-5

Figure Lengend Snippet: Fig. 4 THBS1 suppresses inflammation and immunosurveillance in an OC MTO mouse model. (A) Workflow illustrating the transplantation of ID8 OC cells into wild-type (WT) and THBS1 knockout (KO) mice. (B) IHC analysis of Ki67 and cleaved caspase-3 (C-Cas-3) staining intensities in tumor tissues. (C) Immunofluorescence analysis of VEGFA expression in tumor tissues. (D) IHC analysis of CD206 staining intensity in tumor tissues. (E–F) IHC analysis of CD4 + and CD8 + T cell infiltration in tumor tissues. (G–H) IHC analysis of PD-L1 and GAL-3 staining intensities in tumor tissues. Data are presented as violin plots, with each point representing a single mouse. Statistical significance was determined using two-way ANOVA, followed by Tukey’s multiple comparison test (P < 0.05)

Article Snippet: Recombinant THBS1 protein (R&D Systems) was added to the culture at varying concentrations (0–100 ng/mL).

Techniques: Transplantation Assay, Knock-Out, Staining, Immunofluorescence, Expressing, Comparison

Journal: Journal of ovarian research

Article Title: Thrombospondin-1 induces CD8 + T cell exhaustion and immune suppression within the tumor microenvironment of ovarian cancer.

doi: 10.1186/s13048-025-01668-5

Figure Lengend Snippet: Fig. 5 THBS1 knockdown (KD) alleviates T cell exhaustion. (A) Immunofluorescence analysis of CD8+/CTLA4 + and CD8+/VISTA + T cell populations in tumor tissues. (B) CD3 + T cells isolated from C57 mice were stimulated with anti-CD3/CD28 to induce polarization into CD8 + T cells, with or without recombinant THBS1 protein. (C) Flow cytometry analysis of CD69+, TCF7+, PFN+, VISTA+, PD-1+, and CTLA4 + CD8 + T cells. (D) ELISA quantification of PFN, GZMB, IFN-γ, and IL-2 levels in culture supernatants. (E) Annexin-V/CFSE staining to evaluate the cytotoxic effect of T cells on ID8 OC cells. Data are presented as violin plots, with each point representing a single mouse. Statistical significance was determined using two-way ANOVA, followed by Tukey’s multiple comparison test (P < 0.05)

Article Snippet: Recombinant THBS1 protein (R&D Systems) was added to the culture at varying concentrations (0–100 ng/mL).

Techniques: Knockdown, Immunofluorescence, Isolation, Recombinant, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining, Comparison

Journal: Journal of ovarian research

Article Title: Thrombospondin-1 induces CD8 + T cell exhaustion and immune suppression within the tumor microenvironment of ovarian cancer.

doi: 10.1186/s13048-025-01668-5

Figure Lengend Snippet: Fig. 6 THBS1 expression is transcriptionally regulated by SNF2H. (A) Prediction of an SNF2H-binding motif near the THBS1 transcription start site using the JASPAR database. (B) Comparison of SNF2H expression levels between OC tissues (TCGA-OV dataset) and normal ovarian tissues (GTEx dataset). (C) Association of SNF2H expression with poor prognosis in OC patients (TCGA-OV dataset). (D) IHC analysis of SNF2H staining intensity in tumor and adja cent normal tissues from 97 OC patients. (E) Spearman correlation analysis of THBS1 and SNF2H staining intensities. (F) Pearson correlation analysis of THBS1 and SNF2H expression levels in the TCGA-OV dataset. (G) qPCR analysis of SNF2H and THBS1 mRNA expression after SNF2H knockdown in SKOV3 and A2780 cells. (H) ChIP analysis with anti-SNF2H showing enrichment of THBS1 promoter fragments. Data are presented as violin plots, with each point representing an experimental replicate or patient sample. Statistical significance was determined using Student’s t-test or one-way ANOVA, followed by Tukey’s multiple comparison test (P < 0.05)

Article Snippet: Recombinant THBS1 protein (R&D Systems) was added to the culture at varying concentrations (0–100 ng/mL).

Techniques: Expressing, Binding Assay, Comparison, Staining, Knockdown