ldn 193189  (Tocris)

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: Other drugs and compounds used in this study included: GW9662 (MCE, HY-16578; intraperitoneal injection, 1 mg/kg body weight/day, administered continuously for 4 weeks), T0070907 (Selleck, S2871; intraperitoneal injection, 2 mg/kg body weight/day, administered continuously for 4 weeks), rapamycin (MCE, HY-10219; subcutaneous injection, 3 mg/kg body weight/day, administered continuously for 4 weeks), Rosiglitazone (MCE, HY-17386; oral gavage, 3 mg/kg body weight/day, administered continuously for 2 weeks), LY294002 (Selleck, S1105; intraosseous injection, 10 μM, 5 μL per dose per week, administered for 1 or 4 weeks), DMH1 (Selleck, S7146; intraperitoneal injection, 5 mg/kg body weight/day, administered continuously for 4 weeks), Noggin (PeproTech, 250-38; intraosseous injection, 50 ng per dose, twice per week, administered for 2 or 4 weeks), LDN-193189 (Selleck, S2618; intraperitoneal injection, 3 mg/kg body weight/day, administered for 1 or 4 weeks), IGF-1 (PeproTech, 250-19; intraosseous injection, 4 μg per dose per week, administered for 2 weeks), IGF-1 neutralizing antibody (R&D Systems, AF-791; intraosseous injection, 2 μg per dose, twice per week, administered for 2 or 4 weeks), VEGF neutralizing antibody (R&D Systems, AF-493-NA; intraosseous injection, 2 μg per dose, twice per week, administered for 4 weeks), PDGF-AA neutralizing antibody (R&D Systems, AF-221-NA; intraosseous injection, 2 μg per dose, twice per week, administered for 4 weeks), PDGF-BB neutralizing antibody (R&D Systems, AF-220-NA; intraosseous injection, 2 μg per dose, twice per week, administered for 4 weeks), TGF-β1 neutralizing antibody (R&D Systems, MAB2401; intraosseous injection, 2 μg per dose, twice per week, administered for 4 weeks), TGF-β2 neutralizing antibody (R&D Systems, AB-112-NA; intraosseous injection, 2 μg per dose, twice per week, administered for 4 weeks).

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