Journal: International Journal of Molecular Medicine

Article Title: TNF-α induces premature senescence in tendon stem cells via the NF-κB and p53/p21/cyclin E/CDK2 signaling pathways

doi: 10.3892/ijmm.2025.5581

Figure Lengend Snippet: Effects of the NF-κB and p53/p21/cyclin E/CDK2 signaling pathways on senescence in TNF-α-treated TSCs. (A) ROS staining of TSCs using DCF fluorescence probe, showing intracellular ROS distribution. Scale bar=100 µ m. (B) Quantitative analysis of DCF fluorescence intensity, demonstrating TNF-α-induced elevation of ROS levels. (C) Immunofluorescence staining of γ-H2A.X. Following stimulations with TNF-α (20 ng/ml, six times), the proportion of γ-H2A.X-positive TSCs exhibited a considerable increase. Scale bar=100 µ m. (D) Quantitative analysis of γ-H2A.X-positive TSCs following TNF-α treatment. (E) Expression of γ-H2A.X, H2A.X, p-p65 and p65 following TNF-α stimulation as assessed by western blot. GAPDH was used as a control. (F) Bar groups showed the relative density of γ-H2A.X, H2A.X, p-p65 and p65. (G) Expression of p53, p21, cyclin E and CDK2 following TNF-α stimulation as assessed by western blot. GAPDH was used as a control. (H) Relative density of p53, p21, cyclin E and CDK2. (I) Immunofluorescence examination of p65, p53 and p21 expression was consistent with western blotting. Scale bar=100 µ m. * P<0.05, ** P<0.01, *** P<0.001, **** P<0.0001. ROS, reactive oxygen species; TSCs, tendon stem cells; p-, phosphorylation; ns, not significant.

Article Snippet: After washing by Tris-Buffered Saline with Tween 20 (TBST), membranes were incubated with secondary antibodies (1:10,000) (cat. no. D110087, D110058, Sangon Biotech) at room temperature for 2 h. Primary antibodies included GAPDH (cat. no. 10494-1-AP, anti-rabbit, Proteintech Group, Inc.), p65 (cat. no. sc-8008, anti-mouse, Santa Cruz Biotechnology), p53 (cat. no. ab26, anti-mouse, Abcam), p21 (cat. no. ab188224, anti-rabbit, Abcam), phosphorylated p65 (p-p65; cat. no. sc-136548, anti-mouse, Santa Cruz Biotechnology), γ-H2A.X (cat. no. ab81299, anti-rabbit, Abcam), CDK2 (cat. no. ab32147, anti-rabbit, Abcam) and cyclin E (cat. no. 11554-1-AP, anti-rabbit, Proteintech Group, Inc.), H2A.X (cat. no. 10856-1-AP, anti-rabbit, Proteintech Group, Inc.).

Techniques: Protein-Protein interactions, Staining, Fluorescence, Immunofluorescence, Expressing, Western Blot, Control, Phospho-proteomics

Journal: International Journal of Molecular Medicine

Article Title: TNF-α induces premature senescence in tendon stem cells via the NF-κB and p53/p21/cyclin E/CDK2 signaling pathways

doi: 10.3892/ijmm.2025.5581

Figure Lengend Snippet: Effects of etanercept on the NF-κB and p53/p21/cyclin E/CDK2 signaling pathways in tendon stem cell senescence. (A) ROS staining of TSCs. ROS generation was markedly elevated following repeated TNF-α stimulation and subsequently reduced after repeated administration of etanercept. Scale bar=100 µ m. (B) Quantitative analysis of DCF fluorescence intensity. (C) Expression of γ-H2A.X, H2A.X, p53, p21, p-p65 and p65 after stimulation with TNF-α + etanercept as assessed by western blotting. GAPDH was used as a control. (D) Bar groups showed the relative density of γ-H2A.X, H2A.X, p53, p21, p-p65 and p65. (E) Immunofluorescence of γ-H2A.X and p65 yielded consistent results with those from western blotting. Scale bar=100 µ m. (F) Immunofluorescence of p53 and p21 yielded consistent results with those from western blotting. Scale bar=100 µ m. . ** P<0.01, *** P<0.001, **** P<0.0001. ROS, reactive oxygen species; p-, phosphorylation; ns, not significant.

Article Snippet: After washing by Tris-Buffered Saline with Tween 20 (TBST), membranes were incubated with secondary antibodies (1:10,000) (cat. no. D110087, D110058, Sangon Biotech) at room temperature for 2 h. Primary antibodies included GAPDH (cat. no. 10494-1-AP, anti-rabbit, Proteintech Group, Inc.), p65 (cat. no. sc-8008, anti-mouse, Santa Cruz Biotechnology), p53 (cat. no. ab26, anti-mouse, Abcam), p21 (cat. no. ab188224, anti-rabbit, Abcam), phosphorylated p65 (p-p65; cat. no. sc-136548, anti-mouse, Santa Cruz Biotechnology), γ-H2A.X (cat. no. ab81299, anti-rabbit, Abcam), CDK2 (cat. no. ab32147, anti-rabbit, Abcam) and cyclin E (cat. no. 11554-1-AP, anti-rabbit, Proteintech Group, Inc.), H2A.X (cat. no. 10856-1-AP, anti-rabbit, Proteintech Group, Inc.).

Techniques: Protein-Protein interactions, Staining, Fluorescence, Expressing, Western Blot, Control, Immunofluorescence, Phospho-proteomics

Journal: International Journal of Molecular Medicine

Article Title: TNF-α induces premature senescence in tendon stem cells via the NF-κB and p53/p21/cyclin E/CDK2 signaling pathways

doi: 10.3892/ijmm.2025.5581

Figure Lengend Snippet: Impact of TNF-α on TSCs in normal tendon tissues. Under physiological conditions, TSCs exhibit typical functionality, characterized by regular cell cycles, intact F-actin structures, low levels of ROS and normal expressions of transcription factors. Following stimulation by TNF-α, TSCs experience increased ROS production and DNA damage, activation of the NF-κB signaling pathway (resulting in elevated levels of p-p65 and p65, leading to p65 translocation to the nucleus) and modulation of the p53/p21/cyclin E/CDK2 signaling pathways (resulting in upregulation of p53 and p21 and downregulation of cyclin E and CDK2). These changes induce senescence in TSCs, characterized by alterations such as enlarged cell volume and disrupted F-actin structures. Etanercept, a TNF-α inhibitor, mitigates these effects by binding to TNF-α, thereby inhibiting the activation of signaling pathways. This inhibition leads to reduced ROS levels, mitigates DNA damage, decreases expression of p53, p21 and p-p65, normalizes cyclin E and CDK2 expression and ultimately reverses senescence in TSCs, thereby restoring normal cellular functions. Figure created using BioRender ( app.biorender.com/illustrations ). TSC, tendon stem cells; ROS, reactive oxygen species; F-actin, filamentous-actin; p-, phosphorylation; TNFR, tumor necrosis factor receptor; SA-β-gal, senescence-associated β-galactosidase.

Article Snippet: After washing by Tris-Buffered Saline with Tween 20 (TBST), membranes were incubated with secondary antibodies (1:10,000) (cat. no. D110087, D110058, Sangon Biotech) at room temperature for 2 h. Primary antibodies included GAPDH (cat. no. 10494-1-AP, anti-rabbit, Proteintech Group, Inc.), p65 (cat. no. sc-8008, anti-mouse, Santa Cruz Biotechnology), p53 (cat. no. ab26, anti-mouse, Abcam), p21 (cat. no. ab188224, anti-rabbit, Abcam), phosphorylated p65 (p-p65; cat. no. sc-136548, anti-mouse, Santa Cruz Biotechnology), γ-H2A.X (cat. no. ab81299, anti-rabbit, Abcam), CDK2 (cat. no. ab32147, anti-rabbit, Abcam) and cyclin E (cat. no. 11554-1-AP, anti-rabbit, Proteintech Group, Inc.), H2A.X (cat. no. 10856-1-AP, anti-rabbit, Proteintech Group, Inc.).

Techniques: Activation Assay, Translocation Assay, Protein-Protein interactions, Binding Assay, Inhibition, Expressing, Phospho-proteomics

Journal: Experimental and Therapeutic Medicine

Article Title: Milk-derived exosomes exert anti-inflammatory activity in lipopolysaccharide-induced RAW264.7 cells by modulating the TLR4/NF-κB and PI3K/AKT signaling pathways

doi: 10.3892/etm.2025.12899

Figure Lengend Snippet: Effects of M-Exos on the expression of TLR4/NF-κB signaling pathway related proteins in RAW264.7 cells. (A) Western blot analysis of TLR4/NF-κB signaling pathway-related proteins. (B) Protein semi-quantitative analysis of TLR4 protein. (C) Protein semi-quantitative analysis of p-P65 protein. (D) Protein semi-quantitative analysis of p-P65/P65. (E) Protein semi-quantitative analysis of p-iκBα protein. (F) Protein semi-quantitative analysis of p-iκBα/iκBα. Data are presented as mean ± SEM (n=3). * P<0.05, ** P<0.01 and *** P<0.001 vs. control group. # P<0.05, ## P<0.01 and ### P<0.001 vs. LPS group. M-Exos, milk-derived exosomes; TLR4, toll-like receptor 4; LPS, lipopolysaccharides; Con, control.

Article Snippet: Rabbit anti-phosphorylated (p)-p65, anti-p65 antibodies and HRP-conjugated goat anti-rabbit IgG were purchased from Sangon Biotech.

Techniques: Expressing, Western Blot, Control, Derivative Assay