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Journal: Advanced Science
Article Title: Bone Marrow Macrophage STING Signaling is a Direct Nexus Linking Marrow Adipose Expansion to NAD + Depletion and Osteogenic Decline in Skeletal Aging
doi: 10.1002/advs.77571
Figure Lengend Snippet: BMAd‐derived sDPP4 activates BMM STING signaling through TLR4 binding and mtDNA cooperation. (a) Schematic diagram of sDPP4‐mediated STING activation. (b) Representative immunoblots of TLR2 and TLR4 protein levels in BMMs from rDPP4‐treated or SN‐rDPP4‐treated male mice ( n = 3 biological replicates). (c) Representative immunoblots of TAK1 and STING protein/phosphorylation levels in BMMs stimulated with SN‐rDPP4 and treated with C29 or Resatorvid ( n = 3 biological replicates). (d) Molecular docking analysis of the direct binding mode between the TLR4/MD‐2 complex and the DPP4 structure. (e) SPR analysis of the direct interaction between rDPP4 and rTLR4. (f) Co‐Immunoprecipitation analysis of Flag‐ Dpp4 and HA‐ Tlr4 binding. (g) Co‐Immunoprecipitation analysis of DPP4 deletion mutants binding to full‐length TLR4. (h) qPCR analysis of cytosolic DNA extracted from the cytosolic extracts of 3 M‐/24 M‐BMMs ( n = 6 biological replicates). (i,j) Representative immunofluorescence staining of s anti‐dsDNA (green), anti‐Hsp60 (red), and DAPI (blue) in 3 M‐/18 M BMMs (scale bar, 1 µm) (i). The percentage of dsDNA (green) outside of mitochondria (red) was quantified (j) ( n = 15 from 3 biological replicates). (k) Representative immunoblots of cGAS, STING, TBK1, IRF3, NF‐κB, and IκB protein/phosphorylation levels in BMMs treated with EtBr ( n = 3 biological replicates). (l) Flow cytometry analysis and quantification of mtROS production in WT‐3 M‐/WT‐18 M‐BMMs and Sting ΔMφ ‐18 M‐BMMs ( n = 6 biological replicates). (m) Flow cytometry analysis and quantification of mtROS production in SN‐rDPP4‐treated or co‐cultured BMMs with or without Cgas and Sting knockdown ( n = 6 biological replicates). Data are shown as mean ± SD.
Article Snippet: The
Techniques: Derivative Assay, Binding Assay, Activation Assay, Western Blot, Phospho-proteomics, Docking Assay, Analysis, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Flow Cytometry, Cell Culture, Knockdown