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Experimental validation of bioinformatically identified inflammatory cytokines, ECM-related, and calcification-regulatory genes. (A) Heatmap illustrating genes involved in regulating osteoblast growth. (B) PPI network of calcification-regulatory genes from BP terms, highlighting hub genes such as Igf1, Tgfb3, and MMP-9. (C) PPI network of ECM-regulatory genes derived from CC terms, highlighting hub genes such as Igf1, <t>Wnt5a,</t> and Spp1. (D) Western blot analysis and quantitative analysis of COX-2/β-actin, MMP-9/β-actin, Wnt5a/β-actin, Spp1/β-actin, Tgfb3/β-actin and Igf1/β-actin ratios in thrombin- and PD03-treated osteoblasts. (E) The ratios of COX-2/β-actin, MMP-9/β-actin, Wnt5a/β-actin, Spp1/β-actin, Tgfb3/β-actin and Igf1/β-actin were compared among different groups. Data are presented as mean ± SD (n = 3). P-values were determined by one-way ANOVA (multi-group comparisons) (*p < 0.05; **p < 0.01; ***p < 0.001; ns, P >0.05). Scale bar: 100 μm.
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Experimental validation of bioinformatically identified inflammatory cytokines, ECM-related, and calcification-regulatory genes. (A) Heatmap illustrating genes involved in regulating osteoblast growth. (B) PPI network of calcification-regulatory genes from BP terms, highlighting hub genes such as Igf1, Tgfb3, and MMP-9. (C) PPI network of ECM-regulatory genes derived from CC terms, highlighting hub genes such as Igf1, <t>Wnt5a,</t> and Spp1. (D) Western blot analysis and quantitative analysis of COX-2/β-actin, MMP-9/β-actin, Wnt5a/β-actin, Spp1/β-actin, Tgfb3/β-actin and Igf1/β-actin ratios in thrombin- and PD03-treated osteoblasts. (E) The ratios of COX-2/β-actin, MMP-9/β-actin, Wnt5a/β-actin, Spp1/β-actin, Tgfb3/β-actin and Igf1/β-actin were compared among different groups. Data are presented as mean ± SD (n = 3). P-values were determined by one-way ANOVA (multi-group comparisons) (*p < 0.05; **p < 0.01; ***p < 0.001; ns, P >0.05). Scale bar: 100 μm.
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Experimental validation of bioinformatically identified inflammatory cytokines, ECM-related, and calcification-regulatory genes. (A) Heatmap illustrating genes involved in regulating osteoblast growth. (B) PPI network of calcification-regulatory genes from BP terms, highlighting hub genes such as Igf1, Tgfb3, and MMP-9. (C) PPI network of ECM-regulatory genes derived from CC terms, highlighting hub genes such as Igf1, Wnt5a, and Spp1. (D) Western blot analysis and quantitative analysis of COX-2/β-actin, MMP-9/β-actin, Wnt5a/β-actin, Spp1/β-actin, Tgfb3/β-actin and Igf1/β-actin ratios in thrombin- and PD03-treated osteoblasts. (E) The ratios of COX-2/β-actin, MMP-9/β-actin, Wnt5a/β-actin, Spp1/β-actin, Tgfb3/β-actin and Igf1/β-actin were compared among different groups. Data are presented as mean ± SD (n = 3). P-values were determined by one-way ANOVA (multi-group comparisons) (*p < 0.05; **p < 0.01; ***p < 0.001; ns, P >0.05). Scale bar: 100 μm.

Journal: Frontiers in Immunology

Article Title: PD0325901 alleviates thrombin-inhibited osteogenic differentiation through an IL-1β-activated feedback loop between MEK-Erk1/2 and NF-κB signal pathways: insights from bioinformatics and experimental verification

doi: 10.3389/fimmu.2026.1730337

Figure Lengend Snippet: Experimental validation of bioinformatically identified inflammatory cytokines, ECM-related, and calcification-regulatory genes. (A) Heatmap illustrating genes involved in regulating osteoblast growth. (B) PPI network of calcification-regulatory genes from BP terms, highlighting hub genes such as Igf1, Tgfb3, and MMP-9. (C) PPI network of ECM-regulatory genes derived from CC terms, highlighting hub genes such as Igf1, Wnt5a, and Spp1. (D) Western blot analysis and quantitative analysis of COX-2/β-actin, MMP-9/β-actin, Wnt5a/β-actin, Spp1/β-actin, Tgfb3/β-actin and Igf1/β-actin ratios in thrombin- and PD03-treated osteoblasts. (E) The ratios of COX-2/β-actin, MMP-9/β-actin, Wnt5a/β-actin, Spp1/β-actin, Tgfb3/β-actin and Igf1/β-actin were compared among different groups. Data are presented as mean ± SD (n = 3). P-values were determined by one-way ANOVA (multi-group comparisons) (*p < 0.05; **p < 0.01; ***p < 0.001; ns, P >0.05). Scale bar: 100 μm.

Article Snippet: The following antibodies were used in this study: β-actin (AF5001, Beyotime, China), Col1α1 (ab270993, Abcam, UK), Runx2 (AF2593, Beyotime, China), OCN (AF6297, Beyotime, China), Igf1 (AF7179, Beyotime, China), Wnt5a (29793-1-AP, Proteintech, USA), Tgfb3 (AF8142, Beyotime, China), Spp1 (A5427, Bimake, USA), COX-2 (F0327, Bimake, USA), Matrix Metalloproteinase-9 (MMP-9, AF5234, Beyotime, China), Minichromosome Maintenance Complex Component 2 (MCM2, A5172, Bimake, USA), Proliferating Cell Nuclear Antigen (PCNA, SC-25280, Santa, USA), PAR-1 (AF6837, Beyotime, China), IL-1RA (AF7218, Beyotime, China), p65 (8242S, CST, USA), p-p65 (3033T, CST, USA), Erk1/2 (4695T, CST, USA), p-Erk1/2 (4370S, CST, USA), p-Stat3 (Y705) (9145S, CST, USA), p-Stat3 (S727) (9134S, CST, USA), and Stat3 (4904T, CST, USA).

Techniques: Biomarker Discovery, Derivative Assay, Western Blot