Journal: Frontiers in Immunology

Article Title: Exploratory transcriptomic analysis of mouse articular cartilage in response to tissue inhibitor of metalloproteinase 3 identifies inflammation-associated gene expression changes

doi: 10.3389/fimmu.2026.1794078

Figure Lengend Snippet: Experimental design. Femoral head articular cartilage samples from 16 mice were harvested in pairs and each pair was placed in one well of a 96-well plate with serum-free Opti-MEM supplemented with 1% penicillin-streptomycin, maintained under standard normoxic conditions (21% O 2 , 5% CO 2 , 37°C). After three days, half of the cultures were transferred to a hypoxia incubator (3% O 2 ). Following 24 hours of normoxic or hypoxic incubation, cultures were treated with recombinant human TIMP-3 (100 nM; 2.6 µg/ml) or vehicle for 20 hours, and RNA was extracted for RNA sequencing (n = 4 per group). Group labels: NC, normoxia control; NT, normoxia TIMP-3; HC, hypoxia control; HT, hypoxia TIMP-3.

Article Snippet: After 24 hours, cultures were treated with recombinant human TIMP-3 (R&D Systems, Abingdon, Oxon, UK) at 100 nM, (2.6 μg/ml) or vehicle control for 20 hours under normoxia or hypoxia.

Techniques: Incubation, Recombinant, RNA Sequencing, Control

Journal: Frontiers in Immunology

Article Title: Exploratory transcriptomic analysis of mouse articular cartilage in response to tissue inhibitor of metalloproteinase 3 identifies inflammation-associated gene expression changes

doi: 10.3389/fimmu.2026.1794078

Figure Lengend Snippet: TIMP-3 upregulates Saa3 gene expression in cartilage under normoxia and hypoxia. Articular cartilage explants were cultured and processed for RNA-seq as described in the legend to Table 2 . (A) Venn diagram showing overlap of genes differentially regulated in response to TIMP-3 under normoxia (Norm; 21% O 2 ) and hypoxia (Hyp; 3% O 2 ). (B) RT-qPCR validation of Saa3 . RNA-seq (left) and RT-qPCR (right) data shown as log 2 FC versus the mean of the controls (mean ± SD, n = 4). RNA-seq: log 2 FC were calculated from normalized CPM values; § FDR < 0.05 (hypoxia effect), # P < 0.01 (TIMP-3 effect) by quasi-likelihood F-test applied to raw counts (edgeR). RT-qPCR: *P < 0.05 by unpaired two-sided Welch’s t-test on log 2 FC values (-ΔΔCq). Ctrl, control; FC, fold change.

Article Snippet: After 24 hours, cultures were treated with recombinant human TIMP-3 (R&D Systems, Abingdon, Oxon, UK) at 100 nM, (2.6 μg/ml) or vehicle control for 20 hours under normoxia or hypoxia.

Techniques: Gene Expression, Cell Culture, RNA Sequencing, Quantitative RT-PCR, Biomarker Discovery, Control

Journal: Frontiers in Immunology

Article Title: Exploratory transcriptomic analysis of mouse articular cartilage in response to tissue inhibitor of metalloproteinase 3 identifies inflammation-associated gene expression changes

doi: 10.3389/fimmu.2026.1794078

Figure Lengend Snippet: RNA-seq analysis of TIMP-3–treated cartilage under normoxia. (A) MA plot showing differential expression between TIMP-3–treated and control samples, with log 2 FC plotted against average log 2 CPM expression (n = 4). Grey: no change, red: upregulated, blue: downregulated transcripts (P < 0.01 by quasi-likelihood F-test in edgeR, |log 2 FC| > 0.58). The 10 most highly expressed regulated genes are labelled. Complete gene lists are in . (B) Heatmap of differentially expressed genes (log 2 FC versus the mean of the controls). Genes are ordered by hierarchical clustering. Sample labels: NC1-4: normoxia controls; NT1-4: normoxia TIMP-3–treated. (C) Sole significantly enriched pathway (FDR < 0.05) from DAVID analysis of 26 upregulated genes. As only one KEGG pathway passed the significance threshold, it is shown individually together with its enrichment score (Fold enrichment) and FDR for visual consistency within the multipanel figure. (D) RT-qPCR validation. RNA-seq (left) and RT-qPCR (right) data shown as log 2 FC versus the mean of the controls (mean ± SD, n = 4). RNA-seq: log 2 FC were calculated from normalized CPM values; § FDR < 0.05 (hypoxia effect), # P < 0.01 (TIMP-3 effect) by quasi-likelihood F-test applied to raw counts (edgeR). RT-qPCR: *P < 0.05, **P < 0.01, ***P < 0.001 by unpaired two-sided Welch’s t-test on log 2 FC values (-ΔΔCq). CPM, counts per million; Ctrl, control; FC, fold change; Norm, normoxia; Hyp, hypoxia.

Article Snippet: After 24 hours, cultures were treated with recombinant human TIMP-3 (R&D Systems, Abingdon, Oxon, UK) at 100 nM, (2.6 μg/ml) or vehicle control for 20 hours under normoxia or hypoxia.

Techniques: RNA Sequencing, Quantitative Proteomics, Control, Expressing, Quantitative RT-PCR, Biomarker Discovery

Journal: Frontiers in Immunology

Article Title: Exploratory transcriptomic analysis of mouse articular cartilage in response to tissue inhibitor of metalloproteinase 3 identifies inflammation-associated gene expression changes

doi: 10.3389/fimmu.2026.1794078

Figure Lengend Snippet: RNA-seq analysis of TIMP-3–treated cartilage under hypoxia. (A) MA plot showing differential expression between TIMP-3–treated and control articular cartilage, with log 2 FC plotted against average log 2 CPM expression (n = 4). Grey: no change, red: upregulated, blue: downregulated (P < 0.01, |log 2 FC| > 0.58). The 10 most highly expressed regulated genes are labelled. G1 and G2 correspond to E430024I08Rik and AC127578.1 respectively. Complete gene lists are in . (B) Heatmap of differentially expressed genes (log 2 FC versus the mean of the controls). Genes are ordered by hierarchical clustering. Sample labels: HC1-4: hypoxia controls; HT1-4: hypoxia TIMP-3-treated. (C) Protein–protein interaction (PPI) network corresponding to genes downregulated by TIMP-3 under hypoxia (P <0.01 by quasi-likelihood F-test in edgeR, |log 2 FC| > 0.58), generated using STRING (default interaction score ≥ 0.400). All nodes represent the initially filtered gene list and are included to show the network context and highlight that only Pbk and Racgap1 display a documented interaction. Line colors indicate evidence type: green, text mining; pink, experimental; black, co-expression. Combined interaction score: 0.711. (D) RT-qPCR validation. RNA-seq (left) and RT-qPCR (right) data shown as log 2 FC versus the mean of the controls (mean ± SD, n = 4). RNA-seq: log 2 FC were calculated from normalized CPM values; § FDR < 0.05 (hypoxia effect), # P < 0.01 (TIMP-3 effect) by quasi-likelihood F-test applied to raw counts (edgeR); RT-qPCR: *P < 0.05, **P < 0.01 by unpaired two-sided Welch’s t-test on log 2 FC values (-ΔΔCq). CPM, counts per million; Ctrl, control.

Article Snippet: After 24 hours, cultures were treated with recombinant human TIMP-3 (R&D Systems, Abingdon, Oxon, UK) at 100 nM, (2.6 μg/ml) or vehicle control for 20 hours under normoxia or hypoxia.

Techniques: RNA Sequencing, Quantitative Proteomics, Control, Expressing, Generated, Quantitative RT-PCR, Biomarker Discovery