sa β gal staining kit  (Beyotime)

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS attenuates full-blown bone marrow senescence during GC-induced skeletal degeneration. ( A ) Schematic illustration of the experimental design for assessing bone marrow senescence at 4 weeks after combined SCS and MPS treatment. ( B ) Representative images of SA-β-Gal–positive cells (green) in femur after MPS treatment. BM indicates bone marrow; TBM indicates trabecular bone matrix. (Scale bars, 100 μm and 25 μm) ( C – E ) Representative immunofluorescence images at week 4 showing Emcn + sinusoidal ECs, ALP + osteoblasts, and p16 + senescent cells (C), with corresponding quantification of Emcn + p16 + (D) and ALP + p16 + cells (E). n = 6 biological replicates. (Scale bars, 100 μm and 50 μm) ( F – H ) Flow cytometry analysis of CD45 − Ter119 − CD31 + arteriolar ECs in the femur after PBS or SCS treatment (F). Ki-67 + proliferative status was further analyzed within this population (G), and corresponding double-positive cell quantification is shown in (H). n = 6 biological replicates. ( I – K ) Representative flow cytometry plots of CD45 − Ter119 − CD31 − leptin receptor + (LepR + ) mesenchymal stem cells (MSCs) in the bone marrow at 4 weeks (I), with analysis of the proportion of SA-β-Gal–positive cells (J) and corresponding quantification (K). n = 6 biological replicates. ( L ) Representative flow cytometry plots of CD45 − Ter119 − CD144 + cells (including endothelial cells and endothelial progenitors) in the bone marrow at week 4 post-MPS treatment. ( M and N ) Gating and analysis of CD45 − Ter119 − CD144 + HMGB1 + ECs by flow cytometry (M), and corresponding quantification (N). n = 6 biological replicates. ( O and P ) Representative immunofluorescence images showing OPN + osteoblasts and γ-H2A.X + DNA damage marker–positive cells in the femur at 4 weeks (O), with quantification of senescent osteoblasts (P). n = 6 biological replicates. (Scale bars, 100 μm and 50 μm) Data are presented as mean ± SD. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001. Statistical significance was determined using an unpaired two-tailed Student's t -test ( D, E, H, K, N and P ).

Article Snippet: To assess bone marrow senescence at 4 weeks post-SCS treatment, frozen femoral sections were stained with a SA-β-Gal staining kit (Cell Signaling Technology, 9860) according to the manufacturer's protocol.

Techniques: Immunofluorescence, Flow Cytometry, Marker, Two Tailed Test

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS suppresses senescence cascade amplification by attenuating secondary spread from GC-induced primary senescent adipocytes. ( A ) Schematic illustration of SCS intervention exclusively during the fully developed senescent phase of MPS-induced bone marrow. ( B ) qPCR analysis of senescence-associated markers ( Cdkn1b , Cdkn1a , and Cdkn2c ) in bone tissues at 4 weeks following combined SCS and MPS treatment. n = 3 biological replicates. ( C ) ELISA analysis of bone marrow senescence-associated factors (IL-1β, IL-18, TNF-α, IL-6, CXCL1, and CCL3) after 4 weeks of combined treatment with SCS and MPS. n = 4 biological replicates. ( D ) Quantification of the maximal compressive load of the isolated distal femur and femoral diaphysis. n = 6 biological replicates. ( E ) Schematic diagram depicting isolation of bone marrow adipocytes from mice treated with SCS and MPS for 14 days using mature adipocyte-specific fast centrifugation and construction of a senescence propagation model in vitro . ( F and G ) Representative flow cytometry plots (D) and quantification (E) of EdU-positive (proliferating) CD45 − Ter119 − CD31 − LepR + MSCs cultured for 3 days with adipocyte conditioned medium (CM). n = 6 biological replicates. ( H and I ) Representative ALP staining images (F) and corresponding quantification of ALP activity (G) in CD45 − Ter119 − CD31 − LepR + MSCs cultured with SCS-induced adipocyte CM. n = 6 biological replicates. (Scale bars, 50 μm and 30 μm) ( J and K ) Representative Oil Red O staining (H) and quantification (I) of adipogenic differentiation in MSCs cultured with SCS-induced adipocyte CM. n = 6 biological replicates. (Scale bars, 50 μm and 25 μm) ( L and M ) Representative images (J) and quantification (K) of crystal violet-stained fibroblast colony-forming units (CFU-F) in MSCs cultured with various adipocyte CMs. n = 6 biological replicates. (Scale bars, 400 μm) ( N ) qPCR analysis of senescence-related markers ( Cdkn2a and Cdkn1a ) in MSCs treated with different adipocyte CMs. n = 3 biological replicates. ( O and P ) Representative immunofluorescence-FISH images (M) and quantification (N) showing colocalization of γ-H2A.X with telomere-associated foci (TAF) in MSCs cultured with different adipocyte CMs. n = 6 biological replicates. (Scale bars, 7 μm and 1 μm) ( Q and R ) Representative images (O) and quantification (P) of 2D tube formation assays in HUVECs cultured for 3 days with various adipocyte CMs. n = 6 biological replicates. (Scale bars, 100 μm and 25 μm) ( S and T ) Representative images (Q) and quantification (R) of SA-β-Gal–positive HUVECs (green) following 3-day treatment with different adipocyte CMs. n = 6 biological replicates. (Scale bars, 100 μm and 25 μm) ( U ) qPCR analysis of the senescence-related gene LMNB1 in HUVECs treated with various adipocyte CMs. n = 3 biological replicates. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using an unpaired two-tailed Student's t -test ( B, C, D, G, I, K, M, N, R, T and U ).

Article Snippet: To assess bone marrow senescence at 4 weeks post-SCS treatment, frozen femoral sections were stained with a SA-β-Gal staining kit (Cell Signaling Technology, 9860) according to the manufacturer's protocol.

Techniques: Amplification, Enzyme-linked Immunosorbent Assay, Isolation, Centrifugation, In Vitro, Flow Cytometry, Cell Culture, Staining, Activity Assay, Immunofluorescence, Two Tailed Test

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: SCS modulates mesenchymal stem cell lineage bias via activation of the IGF-1/PI3K/Akt/mTOR signaling pathway. ( A ) Quantitative analysis of osteocyte morphology in the trabecular bone matrix of the bone marrow at week 6 after MPS treatment with or without SCS, in the presence of various neutralizing antibodies (NAbs) and antagonistic proteins. ( B ) ELISA analysis of IGF-1 and BMP-2 levels in the femoral bone marrow and peripheral serum at day 7 following SCS treatment under MPS conditions. ( C and D ) Western blot analysis of phospho-PI3K, phospho-Akt, and phospho-mTOR (C), as well as phospho-Smad1/5/8, phospho-ERK, and phospho-p38 (D), in CD45 − Ter119 − CD31 − LepR + MSCs after 15-min stimulation with conditioned medium (CM) derived from bone marrow fluid at day 7 following SCS treatment. ( E – G ) Representative flow cytometry plots (E, F) and quantitative analysis (G) of CD45 − CD31 − Sca-1 + CD24 − adipocyte progenitor cells (APCs), CD45 − CD31 − Sca-1 + CD24 + MSCs (E), and CD45 − CD31 − Sca-1 − PDGFRα + (Pα + ) osteoprogenitor cells (OPCs) (F) from femoral bone marrow at day 14 post-MPS induction with or without combined treatment using SCS and IGF-1 NAb or Noggin. ( H and I ) Representative SA-β-Gal staining images (green) of the femur (H), and corresponding quantification (I), at week 4 following MPS treatment with SCS in combination with IGF-1 NAb or DMH1. Insets show magnified views of bone marrow (BM) and trabecular bone matrix (TBM) regions. (Scale bars, 100 μm and 25 μm) ( J ) qPCR analysis of 12 senescence-associated markers in ex vivo femoral bone tissues at week 4 following MPS treatment with SCS in combination with IGF-1 NAb or DMH1. ( K ) Representative Oil Red O staining images of CD45 − Ter119 − CD31 − LepR + MSCs sorted from femurs at day 7 following MPS treatment with SCS in combination with LY294002 or LDN-193189, after in vitro adipogenic induction. (Scale bars, 50 μm and 25 μm) ( L and M ) γ-H2A.X and telomere-associated DNA damage foci (TAFs) co-localization analysis (L), and corresponding quantification (M), in CD45 − Ter119 − CD31 + arteriolar ECs sorted from femurs at day 28 following MPS treatment with SCS in combination with rapamycin or LDN-193189, using immuno-FISH staining. (Scale bars, 7 μm and 1 μm) ( N and O ) Sequential fluorescent labeling using calcein (N) and quantification of mineral apposition rate (O) in femurs treated with SCS and MPS for 4 weeks, with or without LY294002 and/or GW9662. (Scale bars, 50 μm) ( P ) ELISA analysis of five senescence-associated cytokines in femoral bone marrow at day 28 following MPS treatment with SCS in combination with rapamycin and/or T0070907. ( Q and R ) Representative t-distributed stochastic neighbor embedding (t-SNE) plots (Q) from flow cytometric analysis of CD45 − CD31 − Sca-1 + CD24 − APCs, CD45 − CD31 − Sca-1 + CD24 + MSCs, CD45 − CD31 − Sca-1 − Pα + OPCs, CD45 − Ter119 − CD31 + arteriolar ECs, and CD45 − Ter119 − Emcn + sinusoidal ECs at day 14 following MPS treatment with SCS in combination with IGF-1 and/or rosiglitazone, and quantitative analysis of APCs (R) ( S ) Heatmap showing the fluorescent intensity distribution of Lamin-B1 expression across five cellular subpopulations as identified in the t-SNE clustering plot. ∗ P < 0.05 vs. IgG (empty lacunae); # P < 0.05 vs. IgG (filled lacunae). ∗ P < 0.05 vs. SCS; # P < 0.05 vs. SCS + IGF-1 NAb. Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using an unpaired two-tailed Student's t -test ( B ), or one-way ANOVA with Tukey's post hoc test ( A, G, I, J, O, P and R ).

Article Snippet: To assess bone marrow senescence at 4 weeks post-SCS treatment, frozen femoral sections were stained with a SA-β-Gal staining kit (Cell Signaling Technology, 9860) according to the manufacturer's protocol.

Techniques: Activation Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Derivative Assay, Flow Cytometry, Staining, Ex Vivo, In Vitro, Labeling, Expressing, Two Tailed Test

Journal: Bioactive Materials

Article Title: Sulfated polysaccharide prevents senescent adipocyte-driven osteonecrosis by stem cell fate reprogramming

doi: 10.1016/j.bioactmat.2025.11.039

Figure Lengend Snippet: Comparative analysis of SCS and D + Q drugs on glucocorticoid-induced bone marrow senescence inhibition. ( A ) Schematic diagram showing the treatment of SCS and D + Q after glucocorticoid-induced senescence. ( B and C ) Representative flow cytometry images of bone marrow SA-β-Gal for senescence detection on day 42 (B), with corresponding quantification analysis (C). n = 6 biological replicates. ( D and E ) ELISA detection of TNF-α and IL-1β levels in bone marrow supernatant. n = 6 biological replicates. ( F ) Schematic diagram of SCS and D + Q treatment in the early stage of glucocorticoid-induced senescence. ( G and H ) Representative flow cytometry images of p16-positive senescent cells in bone marrow on day 42 (G), with corresponding quantification analysis (H). n = 6 biological replicates. ( I and J ) ELISA detection of TNF-α and IL-1β levels in bone marrow supernatant. n = 6 biological replicates. ( K-M ) Representative images of HE staining of the distal femur with macro and high-magnification images (K), and quantification of trabecular and cortical bone empty lacunae (L and M). n = 6 biological replicates. (Scale bars, 550 μm and 25 μm) ( N and O ) Representative ALP staining images of in vitro osteogenic differentiation of bone marrow LepR + MSCs after 14 days (N), with corresponding quantification analysis (O). n = 6 biological replicates. (Scale bars, 50 μm and 25 μm) Data are presented as mean ± SD. ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001; ns, not significant. Statistical significance was determined using one-way ANOVA with Tukey's post hoc test ( C , D , E , H , I , J , L , M and O ).

Article Snippet: To assess bone marrow senescence at 4 weeks post-SCS treatment, frozen femoral sections were stained with a SA-β-Gal staining kit (Cell Signaling Technology, 9860) according to the manufacturer's protocol.

Techniques: Inhibition, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Staining, In Vitro

Journal: Poultry Science

Article Title: miRNA profiling reveals that gga-let-7i/COL1A2 axis induces cell cycle arrest and triggers cellular senescence to accelerate ovarian aging in laying hens by suppressing the PI3K/AKT/MDM2 pathway

doi: 10.1016/j.psj.2026.106542

Figure Lengend Snippet: gga-let-7i promotes the senescence of chicken granulosa cells. (A and B) Functional impact of gga-let-7i overexpression and interference on the relative expression of senescence-related genes, n = 9. (C–E) Overexpression of gga-let-7i upregulates protein levels of the core senescence regulators p53 and p21, while downregulating the senescence inhibitor MDM2. Conversely, gga-let-7i knockdown exhibits the opposite effects, n = 3. (F and G) Effect of gga-let-7i overexpression or knockdown on the proportion of SA-β-gal-positive chicken granulosa cells, n = 3, scale bar = 100 μm. All data were derived from at least three independent replicates and are presented as the mean ± SEM. *, P < 0.05; ⁎⁎ , P < 0.01.

Article Snippet: Senescence of GCs was assessed using the SA-β-gal staining kit (Beyotime) following manufactures’ instructions.

Techniques: Functional Assay, Over Expression, Expressing, Knockdown, Derivative Assay

Journal: Poultry Science

Article Title: miRNA profiling reveals that gga-let-7i/COL1A2 axis induces cell cycle arrest and triggers cellular senescence to accelerate ovarian aging in laying hens by suppressing the PI3K/AKT/MDM2 pathway

doi: 10.1016/j.psj.2026.106542

Figure Lengend Snippet: gga-let-7i bound with COL1A2 to promote GC senescence via suppressing PI3K/AKT/MDM2 signaling pathway. (A) Validation of COL1A2 knockdown efficiency, n = 9. (B) qPCR detected relative proliferation-related gene levels regulated by si-COL1A2, n = 9. (C) Interference of COL1A2 blocked cell cycle of GCs, n = 3. (D and E) COL1A2 inhibition downregulated EdU positive cell ration in GCs, n = 3, scale bar = 200 μm. (F) qPCR detected relative senescence-related gene levels regulated by si-COL1A2, n = 9. (G–I) si-COL1A2 effectively suppresses COL1A2 protein, concomitantly upregulating p53 and downregulating MDM2, and ultimately increasing the proportion of SA-β-gal-positive cells, n = 3, scale bar = 100 μm. (J–M) Western blot assay revealed protein levels of COL1A2, p53, CDK2, p-AKT/AKT and p-MDM2/MDM2 following co-treatment with si-COL1A2, gga-let-7i inhibitor or empty vector, n = 3. All data were derived from at least three independent replicates and are presented as the mean ± SEM. *, P < 0.05; ⁎⁎ , P < 0.01.

Article Snippet: Senescence of GCs was assessed using the SA-β-gal staining kit (Beyotime) following manufactures’ instructions.

Techniques: Biomarker Discovery, Knockdown, Inhibition, Western Blot, Plasmid Preparation, Derivative Assay

Journal: Antioxidants

Article Title: 2-Methoxystypandrone from Polygonum cuspidatum Rejuvenates Senescence by Reducing Mitochondrial ROS

doi: 10.3390/antiox15030357

Figure Lengend Snippet: 2-MS improves senescence-associated phenotypes. Senescent fibroblasts treated with DMSO (0.01%) or 2-MS (4, 8, 12 μM) at 4-day intervals for 12 days. Then, autofluorescence ( A ), senescence-associated β-galactosidase (SA-β-gal) ( B ), p16 ( C ), cellular proliferation ( D ), CXCL12 ( E ), SLIT2 ( F ), COL1A2 ( G ), MMP-1 ( H ), and HYAL1 expression ( I ) were measured. ( A ) Representative flow graphs of autofluorescence. ( B ) + indicates SA-β galactosidase-positive cells. − indicates SA-β galactosidase negative cells. Scale bar: 10 μm. Statistical analysis was performed using one-way ANOVA followed by Bonferroni’s post hoc test, with results considered not significant (n.s.) or significant at ** p < 0.01. Data represent the mean ± S.D., n = 3.

Article Snippet: For SA-β-Gal staining (9860; Cell Signaling Technology, Beverly, MA, USA), the manufacturer’s instructions were adhered to.

Techniques: Expressing