Journal: Journal of Orthopaedic Translation

Article Title: Upregulation of ACSL1 in synovial macrophages promotes lipid peroxidation via the IκB/NF-κB pathway to accelerate osteoarthritis

doi: 10.1016/j.jot.2025.04.016

Figure Lengend Snippet: Supernatant from macrophages affected chondrocyte homeostasis. (A) Toluidine blue (TB) staining and immunofluorescence staining for COL2, ADAMTS5, and MMP13 in cartilage explants from 3-week-old C57BL/6 mice co-cultured with supernatants from shACSL1-BMDMs (shACSL1 transfection followed by addition of LPS stimulation) and control supernatants (shNC transfection with LPS stimulation). n = 6 per group. Scale bar: 50 μm. (B) Quantification of COL2, ADAMTS5, and MMP13 positive cells in (A). (C) TB staining, SA-β-galactosidase (SA-β-Gal) staining, and immunofluorescence staining for γH2AX in primary chondrocytes treated with supernatants from shACSL1 BMDMs and control. n = 6 per group. Scale bar: 20 μm, 200 μm. (D) Quantification of SA-β-Gal positive cells, γH2AX positive cells, and ROS fluorescence intensity in (C). (E) qRT-PCR analysis of mRNA expression levels of Mmp3, Mmp13, Sox9, and Adamts5 in primary chondrocytes treated with supernatants from shACSL1 BMDMs and control. (F) Western blot analysis of senescence markers (P16, P21), degradation markers (ADAMTS5, MMP13), and synthesis markers (SOX9, COL2) in primary chondrocytes treated with supernatants from shACSL1 BMDMs and control. n = 6 per group. (G) Quantification of protein expression levels normalized to GAPDH in (F). ∗P < 0.05, ∗∗P < 0.01, ns not significant. Data are shown as means ± SD. Statistical significance was determined by unpaired Student's t-test for two-group comparisons.

Article Snippet: Antibodies used for western blotting were: mouse anti-iNOS (Santa Cruz Biotechnology, 1:1000, #sc-7271, USA), rabbit anti-ACSL1 (Proteintech, 1:1000, #13989-1-AP, China), rabbit anti-TNFα (Abcam, 1:1000, #ab6671, USA), rabbit anti-MMP13 (Abcam, 1:1000, #ab39012, USA), rabbit anti-p16 (Proteintech, 1:1000, #10883-1-AP, China), rabbit anti-p21 (Proteintech, 1:1000, #10355-1-AP, China), rabbit anti-SOX9 (Abcam, 1:1000, #ab185966, USA), rabbit anti-p65 (Cell Signaling Technology, 1:1000, #8242, USA), rabbit anti-IκBα (Cell Signaling Technology, 1:1000, #9242, USA), and species-matched horseradish peroxidase-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA).

Techniques: Staining, Immunofluorescence, Cell Culture, Transfection, Control, Fluorescence, Quantitative RT-PCR, Expressing, Western Blot

Journal: bioRxiv

Article Title: Matrix stiffness induces endothelial network senescence

doi: 10.1101/2025.10.05.680536

Figure Lengend Snippet: Endothelial networks cultured in hydrogels of increasing stiffness show elevated expression of senescence markers: (A) CDKN1A and CDKN2A mRNA levels measured by qRT-PCR, (B-C) p21 protein levels assessed by Western blot, and (D-E) p16 protein expression visualized by immunofluorescence staining. Representative confocal images show microvascular networks stained for VE-Cadherin in green, p16 in while, nuclei in blue. Scale bar is 100 µm. N=3. (F-G) SA-β-Gal activity increased with matrix stiffening. Representative confocal images show networks stained for F-actin (red), SA-β-Gal (green), and nuclei (blue). Scale bar: 100 µm. N=3. (H-J) Senescence-associated secretory phenotype (SASP), including cytokines, chemokines, and MMPs analyzed using a Luminex assay. Values were normalized to the amount of dsDNA isolated from each hydrogel construct. N=3. Significance levels were set at ns= not significant (p > 0.05), **p ≤ 0.01, and ****p ≤ 0.0001.

Article Snippet: The constructs were incubated with primary antibodies, including anti rabbit-p16 (1:400, Proteintech) and anti mouse-VE-CAD (1:400, Santa Cruz) overnight at 4 °C.

Techniques: Cell Culture, Expressing, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Activity Assay, Luminex, Isolation, Construct

Journal: bioRxiv

Article Title: Matrix stiffness induces endothelial network senescence

doi: 10.1101/2025.10.05.680536

Figure Lengend Snippet: (A) qRT-PCR analysis revealed significant upregulation of Notch1 receptor, ligands JAG1 and JAG2 , and downstream target HEY1 under stiff conditions. Dll4 ligand gene expression remained unchanged upon matrix stiffening. N=3. (B) Matrix stiffening also induced MAPK8 (encoding JNK1), FOS , and JUN gene expression, key components of the JNK-AP-1 signaling axis, as measured by qRT-PCR. N=3. (C-F) Western blot analysis confirmed increased protein levels of cleaved-Notch1 (NICD), JAG1, and JAG2 in stiffened matrices. GAPDH was used as a protein loading control. N=3. (G-K) Matrix stiffening increased phosphorylation of JNK (p-JNK) in both cytoplasmic and nuclear fractions (N=4), while phosphorylation of cJUN (p-c-JUN) was elevated only in the cytoplasm (N=3). This suggest that nuclear p-c-JUN activation is independent of stiffness-mediated Notch signaling. GAPDH and PCNA were used as protein loading controls for cytoplasm and nucleus, respectively. (L) Treatment with nirogacestat (Niro), a γ-secretase inhibitor, reduced stiffness-induced expression of Notch1 , MAPK8 , FOS , CDKN1A , and CDKN2A (N=3), indicating that Notch signaling regulates JNK-FOS signaling and senescence-associated cell cycle arrest. (M-N) Immunofluorescence staining exhibited reduced p16 expression in nirogacestat-treated microvascular networks compared to untreated stiff controls. Representative confocal images show endothelial networks stained for VE-Cadherin in green, p16 in white, nuclei in blue. Scale bar is 100 µm. N=3. (O) Schematic illustration of the proposed mechanotransductive pathway: matrix stiffening enhances Notch activation, NICD release, which in turn activates the JNK-FOS axis, promoting endothelial senescence and an immunomodulatory phenotype. Significance levels indicated as ns= not significant (p > 0.05), *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.

Article Snippet: The constructs were incubated with primary antibodies, including anti rabbit-p16 (1:400, Proteintech) and anti mouse-VE-CAD (1:400, Santa Cruz) overnight at 4 °C.

Techniques: Quantitative RT-PCR, Gene Expression, Western Blot, Control, Phospho-proteomics, Activation Assay, Expressing, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Matrix stiffness induces endothelial network senescence

doi: 10.1101/2025.10.05.680536

Figure Lengend Snippet: (A) Immunofluorescence staining of fibrotic capsules surrounding surgically explanted synthetic breast implants and adjacent fat tissue showed ECs (CD31) in green, senescence marker (p16 INK4a ) in orange, Notch1 receptor in magenta, nuclei in blue. Representative images of both soft and fibrotic tissues were obtained from the same patient. Scale bar = 100 μm (left), 20 μm (right). (B) Increased p16 expression was observed in ECs within fibrotic capsule regions compared to soft control tissues, including fat and muscle. (C) The proportion of non-senescent ECs expressing Notch1 (CD31 + Notch1 + ) was comparable between soft control and fibrotic tissues, suggesting Notch1 expression in non-senescent ECs is not altered by stiffness. (D) A significantly higher frequency of triple-positive (CD31 + p16 + Notch1 + ) cells in fibrotic regions indicates that Notch 1 expression is enriched specifically within senescent ECs in stiffened environments. (E) Violin plots confirm EC identity through expression of standard EC markers VE-Cadherin (CDH5), PECAM1, and von Willebrand factor (vWF). (F) UMAP projection identifies 132 p16⁺ ECs out of 1,836 total ECs. (G) Volcano plot showing differentially expressed genes between p16⁺ and p16⁻ ECs. Genes meeting adjusted p < 0.05 and log₂ fold change > 0.5 are highlighted. (H) Dot plot displays enrichment of Notch- and JNK-associated genes in senescent ECs. (I) p16⁺ ECs show increased expression of genes related to cell cycle arrest (e.g., CDKN2A/B/C, CCNG1), SASP and inflammatory signaling (e.g., BCL2, BAX, CXCL12/14/16, IL11RA), and ECM remodeling (e.g., MMP3/11/14, PLAUR, SERPINE2, CCN1), supporting their active role in fibrosis-associated tissue remodeling.

Article Snippet: The constructs were incubated with primary antibodies, including anti rabbit-p16 (1:400, Proteintech) and anti mouse-VE-CAD (1:400, Santa Cruz) overnight at 4 °C.

Techniques: Immunofluorescence, Staining, Capsules, Marker, Expressing, Control

Journal: bioRxiv

Article Title: Matrix stiffness induces endothelial network senescence

doi: 10.1101/2025.10.05.680536

Figure Lengend Snippet: Gene Set Enrichment Analysis (GSEA) of p16 + ECs showing enrichment of pathways (adjusted p < 0.05) related to collagen fibril organization, collagen metabolism, and extracellular organization.

Article Snippet: The constructs were incubated with primary antibodies, including anti rabbit-p16 (1:400, Proteintech) and anti mouse-VE-CAD (1:400, Santa Cruz) overnight at 4 °C.

Techniques: