Fig 2: TNC promotes synovial fibroblast activation and migration via activation of the TGF-β/Smad2/3 signaling pathway(A) mRNA expression levels of TBX5, TNC, TGF-β1, Smad2, and Smad3 in human rheumatoid arthritis synovial fibroblasts (RASFs) stimulated with oxidized low-density lipoprotein (ox-LDL).(B) Protein expression levels of TBX5, TNC, TGF-β1, Smad2/3, and phosphorylated Smad2/3 (p-Smad2/3) in RASFs stimulated with ox-LDL, as determined by Western blot.(C) mRNA expression levels of TGF-β1, Smad2, and Smad3 in human synovial fibroblasts overexpressing TNC.(D) Protein expression levels of TGF-β1 and p-Smad2/3 in human synovial fibroblasts overexpressing TNC, as determined by western blot.(E) Immunocytochemical staining of α-SMA, COL-I, and COL-III in RASFs under normal control (NC), ox-LDL stimulation, and TNC overexpression conditions. Scale bars, 50 μm.(F) Representative images from scratch assays showing the migration of human synovial fibroblasts under normal control (NC), ox-LDL stimulation, and TNC overexpression conditions at 0 and 6 h. The bar graph on the right quantifies the migration rate. Scale bars, 100 μm. n = 3 biologically independent experiments. Data are represented as mean ± SD. Statistical analyses were performed using Student’s t tests for two groups (A and B) and one-way ANOVA for multiple comparisons (C, D, and F). ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.

Fig 3: Interaction between TNC and Itgα2 regulates the TGF-β/Smad2/3 signaling pathway and fibroblast behavior(A) Co-immunoprecipitation (CoIP) assay showing the interaction between TNC and Itgα2 in synovial fibroblasts.(B) Reverse CoIP assay confirming the interaction between Itgα2 and TNC in synovial fibroblasts.(C) mRNA expression levels of Itgα2, TGF-β1, Smad2, and Smad3 in synovial fibroblasts with Itgα2 overexpression, Itgα2 knockdown, and negative control.(D) Protein expression levels of Itgα2, TGF-β1, phosphorylated Smad2/3 (p-Smad2/3), and Smad2/3 in synovial fibroblasts with Itgα2 overexpression, Itgα2 knockdown, and negative control, as determined by western blot.(E) Immunocytochemical staining of α-SMA, COL-I, and COL-III in synovial fibroblasts with Itgα2 overexpression, Itgα2 knockdown, and negative control. Scale bars, 50 μm.(F) Representative images from scratch assays showing the migration of synovial fibroblasts with Itgα2 overexpression, Itgα2 knockdown, and negative control at 0 and 6 h. The bar graph on the right quantifies the migration rate. Scale bars, 100 μm.(G) mRNA expression levels of Itgα2, TGF-β1, Smad2, and Smad3 in synovial fibroblasts with Itgα2 overexpression, Itgα2 knockdown, and negative control under ox-LDL stimulation.(H) Protein expression levels of Itgα2, TGF-β1, phosphorylated Smad2/3 (p-Smad2/3), and Smad2/3 in synovial fibroblasts with Itgα2 overexpression, Itgα2 knockdown, and negative control under ox-LDL stimulation, as determined by western blot.(I) Immunocytochemical staining of α-SMA, COL-I, and COL-III in synovial fibroblasts with Itgα2 overexpression, Itgα2 knockdown, and negative control under ox-LDL stimulation. Scale bars, 50 μm.(J) Representative images from scratch assays showing the migration of synovial fibroblasts with Itgα2 overexpression, Itgα2 knockdown, and negative control under ox-LDL stimulation at 0 and 6 h. The bar graph on the right quantifies the migration rate. Scale bars, 100 μm. n = 3 biologically independent experiments. Data are represented as mean ± SD and were analyzed using one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.

Fig 4: In vivo effects of TBX5 modulation on hyperlipidemia-induced frozen shoulder in rats(A) Shoulder joint range of motion in hyperlipidemic (HL) rats with TBX5 overexpression (HL + OE-TBX5) and knockdown (HL+si-TBX5).(B) Representative hematoxylin and eosin (H&E) stained histological sections of shoulder joint tissues from HL, HL + OE-TBX5, and HL+si-TBX5 rats. Scale bars, 50 μm.(C) Representative Masson’s trichrome stained histological sections of shoulder joint tissues from HL, HL + OE-TBX5, and HL+si-TBX5 rats. Scale bars, 50 μm.(D) mRNA expression levels of TBX5, TNC, Itgα2, TGF-β1, Smad2, and Smad3 in shoulder joint tissues from HL, HL + OE-TBX5, and HL+si-TBX5 rats, as determined by qPCR.(E) Protein expression levels of TBX5, TNC, Itgα2, TGF-β1, phosphorylated Smad2/3 (p-Smad2/3), and Smad2/3 in shoulder joint tissues from HL, HL + OE-TBX5, and HL+si-TBX5 rats, as determined by western blot.(F) Immunocytochemical staining of α-SMA, COL-I, and COL-III in shoulder joint tissues from HL, HL + OE-TBX5, and HL+si-TBX5 rats. Scale bars, 50 μm. n = 6 biologically independent rats per group. Data are represented as mean ± SD and were analyzed using one-way ANOVA. ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.

Fig 5: TBX5 regulates TNC and the TGF-β/Smad2/3 signaling pathway(A) Dual-luciferase reporter assay examining the effect of TBX5 overexpression on wild-type and mutant TNC promoter activity. ∗∗p < 0.01, ##p < 0.01.(B) Chromatin immunoprecipitation followed by quantitative PCR (ChIP-qPCR) showing TBX5 binding to different regions of the TNC promoter. ∗∗p < 0.01.(C) Chromatin immunoprecipitation followed by western blot (ChIP-western blot) confirming TBX5 enrichment at the TNC promoter. ∗∗p < 0.01.(D) Quantitative real-time PCR (qPCR) analysis of mRNA expression levels of TNC, TGF-β1, Smad2, and Smad3 in synovial fibroblasts with TBX5 overexpression and knockdown. ∗∗p < 0.01.(E) Western blot analysis of protein expression levels of TNC, TGF-β1, phosphorylated Smad2/3 (p-Smad2/3), and Smad2/3 in synovial fibroblasts with TBX5 overexpression and knockdown. ∗∗p < 0.01.(F) Immunocytochemical staining of α-SMA, COL-I, and COL-III in synovial fibroblasts with TBX5 overexpression and knockdown. Scale bars, 50 μm.(G) Scratch assay showing the migration of synovial fibroblasts with TBX5 overexpression and knockdown. The bar graph on the right quantifies the migration rate. Scale bars, 100 μm ∗∗p < 0.01.(H) Quantitative real-time PCR (qPCR) analysis of mRNA expression levels of TNC, TGF-β1, Smad2, and Smad3 in TBX5-overexpressing synovial fibroblasts with and without TNC knockdown. ∗∗p < 0.01(vs. NC-TBX5 group), ##p < 0.01(vs. OE-TBX5+si-TNC group).(I) Western blot analysis of protein expression levels of TNC, TGF-β1, phosphorylated Smad2/3 (p-Smad2/3), and Smad2/3 in TBX5-overexpressing synovial fibroblasts with and without TNC knockdown. ∗∗p < 0.01(vs. NC-TBX5 group), ##p < 0.01(vs. OE-TBX5+si-TNC group).(J) Immunocytochemical staining of α-SMA, COL-I, and COL-III in synovial fibroblasts under different conditions of TBX5 overexpression and TNC knockdown. Scale bars, 50 μm.(K) Scratch assay showing the migration of synovial fibroblasts under different conditions of TBX5 overexpression and TNC knockdown. The bar graph on the right quantifies the migration rate. Scale bars, 100 μm ∗p < 0.05, ∗∗p < 0.01(vs. NC-TBX5 group), ##p < 0.01(vs. OE-TBX5+si-TNC group). n = 3 biologically independent experiments. Data are represented as mean ± SD. Statistical analyses were performed using one-way ANOVA followed by Tukey’s post hoc test for multiple comparisons.