Journal: Biochemistry and Biophysics Reports

Article Title: Screening of metabolic-related biomarkers linking intervertebral disc degeneration and type 2 diabetes based on comprehensive bioinformatics analysis and machine learning

doi: 10.1016/j.bbrep.2026.102593

Figure Lengend Snippet: BCAA metabolism and immune changes in degenerated NP cells. (A) qRT-PCR was conducted to analysis the mRNA levels of BCAA metabolism-related enzymes and BCAA transporter in grade I/II and grade III/IV NP tissues; n = 20; P < 0.05. (B, C) Expression levels of mRNA for pro-inflammatory factors, BCAA metabolism-related enzymes, and BCAA transporters in control group and TNF-α-treated HNPC cells. (D) Expression of SA-β-gal in HNPC cells stimulated with different concentrations of TNF-α. (E, F) p16, p21, p53 mRNA level in TNF-α-stimulated HNPC cells,and correlation analysis with TNF-α concentration.

Article Snippet: Cellular senescence was assessed using Senescence-Associated β-Galactosidase (SA-β-gal) staining (Senescence β-Galactosidase Staining Kit, #C0602, Beyotime, China), performed according to the manufacturer's protocol.

Techniques: Quantitative RT-PCR, Expressing, Control, Concentration Assay

Journal: Journal of Translational Medicine

Article Title: 3D-Mesenchymal stromal cells derived VASH2 alleviates oxidative stress-induced endothelial senescence by mediating α-tubulin detyrosination in systemic sclerosis

doi: 10.1186/s12967-026-08072-7

Figure Lengend Snippet: 3D-MSCs ameliorated oxidant-induced senescence and restored angiogenic function in HUVECs. ( A ) Representative images of senescence-associated β-galactosidase (SA β-gal) staining in HUVECs after exposure to 200 µM H₂O₂ for 4 h and then co-culture with 2D-MSCs or 3D-MSCs for 24 h. Scale bars, 100 μm. ( B ) Quantification of SA β-gal-positive cells ( n = 6). ( C ) qPCR analysis of senescence markers (p16, p21, p53), LaminB1 (LMNB1), and senescence-associated secretory phenotype (SASP) factors (IL-1α, IL-6, IL-8) ( n = 6). ( D ) Representative western blot images of p16, p21, p53 and LMNB1 proteins. ( E ) Quantitative analysis of protein levels of p16, p21, p53 and LMNB1 ( n = 6). ( F ) Representative immunofluorescence images of p21. Scale bars, 50 μm. ( G ) Representative images of EdU staining. Scale bars, 100 μm. ( H ) Quantification of p21-positive cells ( n = 6). ( I ) Quantification of EdU-positive cells ( n = 6). ( J ) Representative images from transwell migration assays. Scale bars, 200 μm. ( K ) Representative images from tube formation assays. Scale bars, 200 μm. ( L ) Quantification of migrated cells ( n = 6). ( M ) Quantification of junction numbers in HUVEC network ( n = 3). ( N ) Quantification of total segments length in the HUVEC network ( n = 3). ( O ) Representative immunofluorescence images of endothelial junctional proteins (ZO-1 and VE-cadherin). Scale bars, 25 μm. ( P ) Quantitative analysis of ZO-1 fluorescence intensity per cell ( n = 30). ( Q ) Quantitative analysis of VE-cadherin fluorescence intensity per cell ( n = 30). The data were presented as means ± SD. p -values were calculated using ordinary one-way ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001

Article Snippet: HUVECs were fixed with 4% formaldehyde at room temperature for 20 min and then subjected to senescence detection using a β-galactosidase (β-gal) staining kit (Catalog #C0602, Beyotime Biotechnology, Shanghai, China).

Techniques: Staining, Co-Culture Assay, Western Blot, Immunofluorescence, Migration, Fluorescence

Journal: Journal of Translational Medicine

Article Title: 3D-Mesenchymal stromal cells derived VASH2 alleviates oxidative stress-induced endothelial senescence by mediating α-tubulin detyrosination in systemic sclerosis

doi: 10.1186/s12967-026-08072-7

Figure Lengend Snippet: 3D-MSCs rescued senescent HUVECs via VASH2 secretion. ( A ) Heatmap of genes associated with positive regulation of EC proliferation. ( B ) Heatmap of genes related to positive regulation of angiogenesis. ( C ) VASH2 protein levels in conditioned medium from 2D-MSCs, 3D-MSCs, and VASH2-knockdown 3D-MSCs (3D-MSCs si−VASH2 ). ( D ) Representative SA β-gal staining images in HUVECs after exposure to 200 µM H₂O₂ for 4 h and then co-culture with 3D-MSCs or 3D-MSCs si−VASH2 for 24 h. Scale bars, 100 μm. ( E ) Percentage of SA β-gal-positive cells ( n = 6). ( F ) qPCR assessment of senescence markers (p16, p21, p53), LMNB1, and SASP factors (IL-1α, IL-6, IL-8) in HUVECs after co-culture ( n = 6). ( G ) Western blot profiles of senescence-related proteins (p16, p21, p53, LMNB1). ( H ) Representative images corresponding to the quantifications in ( I-N ): p21 immunofluorescence (scale bars, 25 μm), EdU staining (scale bars, 100 μm), transwell migration assay (scale bars, 200 μm), tube formation assay (scale bars, 200 μm), ZO-1 staining (scale bars, 25 μm), and VE-cadherin staining (scale bars, 25 μm). ( I ) Quantification of p21-positive cells ( n = 6). ( J ) Quantification of EdU-positive cells ( n = 6). ( K ) Quantification of migrated cells ( n = 6). ( L ) Quantification of junction numbers in the tube formation assay ( n = 3). ( M ) Quantitative analysis of ZO-1 fluorescence intensity per cell ( n = 30). ( N ) Quantitative analysis of VE-cadherin fluorescence intensity per cell ( n = 30). The data were presented as means ± SD. p -values were calculated using ordinary one-way ANOVA. * p < 0.05, *** p < 0.001, and **** p < 0.0001

Article Snippet: HUVECs were fixed with 4% formaldehyde at room temperature for 20 min and then subjected to senescence detection using a β-galactosidase (β-gal) staining kit (Catalog #C0602, Beyotime Biotechnology, Shanghai, China).

Techniques: Knockdown, Staining, Co-Culture Assay, Western Blot, Immunofluorescence, Transwell Migration Assay, Tube Formation Assay, Fluorescence

Journal: Journal of Translational Medicine

Article Title: 3D-Mesenchymal stromal cells derived VASH2 alleviates oxidative stress-induced endothelial senescence by mediating α-tubulin detyrosination in systemic sclerosis

doi: 10.1186/s12967-026-08072-7

Figure Lengend Snippet: EpoY inhibited the anti-senescence and angiogenic functions of 3D-MSCs by blocking VASH2-mediated α-tubulin detyrosination. ( A ) Immunofluorescence staining of deTyr-Tub (green) and α-tubulin (red) in HUVECs. Scale bars, 50 μm (overview) and 20 μm (magnified). ( B ) Western blot analysis showing protein levels of deTyr-Tub, α-tubulin, VASH2 and senescence markers (p16, p21, p53, LMNB1). ( C ) SA β-gal staining and ( D ) corresponding quantitative analysis of cellular senescence ( n = 6). Scale bars, 100 μm. ( E ) mRNA expression levels of senescence-associated genes (p16, p21, p53, LMNB1, IL-1α, IL-6, IL-8) measured by qPCR. ( F ) Representative images illustrating p21 immunofluorescence (scale bars, 50 μm), EdU proliferation assay (scale bars, 100 μm), transwell migration assay (scale bars, 200 μm), tube formation assay (scale bars, 200 μm), and immunofluorescence staining for ZO-1 and VE-cadherin (scale bars, 25 μm). ( G ) Proportion of p21-positive ECs ( n = 6). ( H ) Percentage of EdU-positive proliferating ECs ( n = 6). ( I ) Number of migrated ECs ( n = 6). ( J ) Junction counts in tube formation assay ( n = 3). ( K ) Total tube length in tube formation assay ( n = 3). ( L ) Mean fluorescence intensity of ZO-1 per cell ( n = 30). ( M ) Mean fluorescence intensity of VE-cadherin per cell ( n = 30). Data were reported as means ± SD. p -values were calculated using one-way ordinary ANOVA. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001

Article Snippet: HUVECs were fixed with 4% formaldehyde at room temperature for 20 min and then subjected to senescence detection using a β-galactosidase (β-gal) staining kit (Catalog #C0602, Beyotime Biotechnology, Shanghai, China).

Techniques: Blocking Assay, Immunofluorescence, Staining, Western Blot, Expressing, Proliferation Assay, Transwell Migration Assay, Tube Formation Assay, Fluorescence

Journal: Journal of Pharmaceutical Analysis

Article Title: Novel bioactive peptides targeting Keap1-Nrf2 interaction for combating UVA-induced skin aging: Computational discovery and experimental validation

doi: 10.1016/j.jpha.2025.101446

Figure Lengend Snippet: Protective effects of Seq1 and Seq3 on ultraviolet A (UVA)-induced cellular aging in human keratinocytes (HaCaT) cells. (A) The effect of peptide treatment on the migration of HaCaT cells. (B) The extent of wound closure was quantified and depicted on a histogram. (C) Assessment of the effects of peptides on senescence associated β-galactosidase (SA-β-Gal) activity in HaCaT cells. (D) Quantitation of SA-β-gal positive cells in HaCaT cells. (E) Immunofluorescence (IF) analysis of phosphorylated γ-H2AX in response to UVA irradiation and peptide treatment at varying concentrations to determine their influence. (F) 2’,7’-Dichlorodihydrofluorescein diacetate (DCFH-DA) staining reveals intracellular reactive oxygen species (ROS) levels in UVA-irradiated HaCaT cells following peptide or tert-butylhydroquinone (t-BHQ) treatment. (G) Fluorescence intensity of ROS. (H) Western blot analysis of Seq1 and Seq3 on the expression of matrix metalloproteinase (MMP)-1 and MMP-9 in UVA-induced HaCat cells. (I) Relative MMP-1 and MMP-9 messenger RNA (mRNA) levels in Seq1 and Seq3 treated UVA-induced HaCat cells. Gene expression was normalized to glyceraldehyde 3-phosphate dehydrogenase ( GAPDH ). (J) Western blot analysis showing the change of inducible nitric oxide synthase (iNOS) and interleukin-1 beta (IL-1β) in HaCat cells. (K) Quantitation of IL-1β and tumor necrosis factor-alpha ( TNF-α ) released by HaCat cells by quantitative real-time polymerase chain reaction (qRT-PCR). (L) Glutathione peroxidase (GSH-Px) activity levels in HaCaT cells. (M) Measurement of superoxide dismutase (SOD) activity levels in HaCaT cells. (N) Nrf2-dependent antioxidant enzymes protein levels for reduced nicotinamide adenine dinucleotide phosphate (NAD(P)H) quinone oxidoreductase 1 (NQO1), heme oxygenase 1 (HO-1), and glutamate-cysteine ligase modifier subunit (GCLM) in UVA-irradiated HaCaT cells treated with peptides. Protein expression normalized to GAPDH. (O) Quantitation of GCLM , HO-1 , and NQO1 released by HaCat cells by qRT-PCR. Unless otherwise indicated in the figure, the peptide concentration was 20 μM. All data are presented as means ± standard deviation (SD) ( n = 3). Statistical significance is denoted by ∗ P < 0.05 , ∗∗ P < 0.01 , ∗∗∗ P < 0.001, and ∗∗∗∗ P < 0.0001 vs. UVA-irradiated group.

Article Snippet: To evaluate cellular senescence induced by peptides and UVA treatments, HaCaT cells were stained using the SA-β-Gal Staining Kit (C0602, Beyotime, Shanghai, China), following the manufacturer's guidelines.

Techniques: Migration, Activity Assay, Quantitation Assay, Immunofluorescence, Irradiation, Staining, Fluorescence, Western Blot, Expressing, Gene Expression, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Concentration Assay, Standard Deviation