tsg101  (Proteintech)

Journal: Bioactive Materials

Article Title: Microenvironment-educated MSC-EVs loaded injectable smart hydrogel for targeting senescent nucleus pulposus cells and inhibiting ferroptosis against intervertebral disc degeneration

doi: 10.1016/j.bioactmat.2026.02.030

Figure Lengend Snippet: Senescent Microenvironment-Educated Mesenchymal Stem Cells Release High-Affinity Senescent NPC Domesticated Extracellular Vesicles. (A) Schematic diagram of the experimental setup for educating MSCs with SASP-CM to generate D-EVs versus N-EVs. (B) Confocal microscopy images showing different EVs internalization by senescent NPCs after 12 h in vitro. (C) Flow cytometry and quantification analysis of different EVs uptake by senescent NPCs. (D) In vivo validation of the senescent niche. Representative fluorescence images following injection of senescence-tracer (Red). (E) In vivo PKH26-labeled D-EVs tracking. (F) Representative SA-β-Gal images and quantification of MSCs treated with SASP-CM or not. (G) Gene Ontology (GO) analysis confirming enrichment of external encapsulating structure organization and cytokine production in Biological Process (BP) categories. (H) Heatmap indicating gene expression associated with EVs biogenesis within D-MSCs and N-MSCs. (I) Heatmap indicating gene expression associated with cytokine production within D-MSCs and N-MSCs. (J and L) Gene Ontology (GO) analysis confirming enrichment of terms related to vesicle organization and transport in the Cellular Component (CC) categories. (K) Western blot analysis confirmed core senescence markers p16 and p21 and DNA damage marker γ-H2AX in N-MSC and D-MSC. (M) Western blot analysis confirmed the expression of CD9, CD63, TSG101, Calnexin, and GM130 in MSC-EVs, N-EVs, or D-EVs. (N) TEM images showing the morphology and size of MSC-derived EVs, N-EVs, and D-EVs. (O) NTA shows size distribution in MSC-EVs, N-EVs, or D-EVs. The data were presented as mean ± SD. n = 3, ns, not significant; ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Article Snippet: After blocked with 5% non-fat milk for 2 h at room temperature, the membranes were incubated with primary antibodies against GAPDH (1:5000, 104941-AP, Proteintech), TSG101 (1:1000, DF8427, Affinity), CD9 (1:1000, AF5139, Affinity), CD63 (1:2000, 25682-1-AP, Proteintech), Calnexin (1:5000, 10427-2-AP, Proteintech), GM130 (1:20000, 11308-1-AP, Proteintech), CXCR3 (1:5000, 26756-1-AP, Proteintech), CXCL10 (1:2000, 10937-1-AP, Proteintech), MMP3 (1:2000, 17873-1-AP, Proteintech), ADAMTS5 (DF13268, Affinity), P16 (AF5484, Affinity), P21 (10355-1-AP, Proteintech), GPX4 (1:1000, 381958, Zen-bio), SLC7A11 (1:1000, 26864-1-AP, Proteintech), ACSL4 (1:5000, 22401-1-AP, Proteintech) and Tubulin (1:10000, T40103 , Abmart) overnight at 4 °C.

Techniques: Confocal Microscopy, In Vitro, Flow Cytometry, In Vivo, Biomarker Discovery, Fluorescence, Injection, Labeling, Gene Expression, Western Blot, Marker, Expressing, Derivative Assay

Journal: Biomedicines

Article Title: Ionic Extracts of Magnesium Powders Promote In Vitro Lymphangiogenesis

doi: 10.3390/biomedicines14040913

Figure Lengend Snippet: Effects of magnesium extracts on lymphangiogenesis-related gene and protein expression in LECs. ( A ) Relative Vegfa mRNA expression levels in LECs treated with Control, Mg (1:10), Mg (1:100), and Mg (1:1000), as determined by qRT-PCR. ( B ) Relative Vegfc mRNA expression levels in LECs under the same treatment conditions. ( C ) Representative Western blot images showing VEGFA and VEGFC protein expression in LECs after treatment with different concentrations of magnesium extracts, with GAPDH used as the internal loading control. ( D ) Additional Western blot analysis showing VEGFR3 protein expression under the same treatment conditions, with β-actin used as the internal loading control. Data in ( A , B ) are presented as the mean ± SD ( n = 3). Statistical analysis for ( A , B ) was performed using one-way ANOVA followed by Tukey’s multiple-comparisons test. Statistical significance is indicated as * p < 0.05, ** p < 0.01; ns, not significant.

Article Snippet: The primary antibodies used in this study included anti-VEGFA rabbit polyclonal antibody (ZEN-BIOSCIENCE Co., Ltd., Chengdu, China), anti-VEGFC rabbit polyclonal antibody (ZEN-BIOSCIENCE Co., Ltd., Chengdu, China), anti-VEGFR3 rabbit polyclonal antibody (Affinity Biosciences, Shanghai, China), anti-GAPDH rabbit polyclonal antibody (Affinity Biosciences, Shanghai, China), and anti-β-actin rabbit polyclonal antibody (Affinity Biosciences, Shanghai, China).

Techniques: Expressing, Control, Quantitative RT-PCR, Western Blot