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 accumulation and sDPP4 release during skeletal aging activate BMM STING signaling. (a) Heatmap of dysregulated factors (≥1.5‐fold change) in the bone marrow supernatant of 3‐/18‐month‐old male rats identified through 4D label‐free technology ( n = 3 biological replicates). (b) Venn diagram of dysregulated factors in the bone marrow of rats. (c) ELISA of sDPP4 levels in conditioned medium collected from BMAds, Pre‐Ocs, BMMs, Ocs, BMSCs, Obs, and Neu ( n = 6 biological replicates; * p < 0.05 compared to BMAds‐CM). (d) Representative immunofluorescence staining showing that DPP4 was markedly detected in BMAds (Perilipin) in femora from 3‐/24‐month‐old male mice (scale bar: 100 µm). (e) ELISA of sDPP4 levels in conditioned medium collected in 3 M‐/18 M‐BMAds ( n = 6 biological replicates). (f,g) Representative micro‐CT images (f) and quantitative analysis of BV/TV, Tb. N, Tb.Th, and Tb.Sp (g) from PBS‐treated or rDPP4‐treated male mice ( n = 6 biological replicates). (h,i) Representative micro‐CT images (h) and quantitative analysis of BV/TV, Tb. N, Tb.Th, and Tb.Sp (i) from AAV9‐BMAd‐shNC‐treated or AAV9‐BMAd‐sh Dpp4 ‐treated male mice ( n = 6 biological replicates). (j) Representative immunoblots of cGAS, STING, TBK1, IRF3, NF‐κB, and IκB protein/phosphorylation levels in BMMs from PBS‐treated or rDPP4‐treated male mice ( n = 3 biological replicates). (k) Representative immunoblots of cGAS and STING protein/phosphorylation levels in BMMs treated with either rDPP4 alone or in combination with 18 M‐SN ( n = 3 biological replicates). Data are shown as mean ± SD.

Article Snippet: Conversely, rDPP4 (Sino Biological, 10688‐H08H) administration involved once‐weekly intramedullary injections (0.2 μg per injection) for 4 weeks.

Techniques: Enzyme-linked Immunosorbent Assay, Immunofluorescence, Staining, Micro-CT, Analysis, Western Blot, Phospho-proteomics

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: Conversely, rDPP4 (Sino Biological, 10688‐H08H) administration involved once‐weekly intramedullary injections (0.2 μg per injection) for 4 weeks.

Techniques: Derivative Assay, Binding Assay, Activation Assay, Western Blot, Phospho-proteomics, Docking Assay, Analysis, Co-Immunoprecipitation Assay, Immunofluorescence, Staining, Flow Cytometry, Cell Culture, Knockdown

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: STING signaling promotes NAD + decline in BMMs during skeletal aging by enhancing Cd38 transcription through IRF3 and P65. (a) WST‐8 analysis of NAD levels in 3, 10, 18, and 24 M‐BMMs ( n = 6 biological replicates). (b) Schematic of key enzymes in the NAD salvage biosynthesis pathway. (c,d) qPCR ( n = 6 biological replicates) and Western blotting ( n = 3 biological replicates) analyses of key enzymes in the NAD + salvage biosynthesis pathway in 3, 10, 18, and 24 M‐BMMs. (e) Fluorometric assay for CD38 NADase activity in 3 M‐/18 M‐BMMs ( n = 6 biological replicates). (f) WST‐8 analysis of NAD + levels in WT‐3 M‐/WT‐18 M‐BMMs and Sting ΔMφ ‐18 M‐BMMs ( n = 6 biological replicates). (g,h) Fluorometric assay and WST‐8 analysis of CD38 NADase activity (g) or NAD + levels (h) in SN‐rDPP4‐treated BMMs with or without Cgas , Sting , Irf3, and Rela knockdown ( n = 6 biological replicates). (i) Representative immunoblots of CD38 protein levels in diABZI‐treated, SN‐rDPP4‐treated, or co‐cultured BMMs with or without Cgas , Sting , Irf3, and Rela knockdown ( n = 3 biological replicates). (j) Dual luciferase reporter assay in HEK 293T cells co‐transfected with CD38 luciferase reporter, IRF3 or P65 plasmid separately, and pRL‐CMV‐Renilla ( n = 6 biological replicates). (k) Chromatin immunoprecipitation assay showing that IRF3 or P65 bound to the CD38 promoter in BMMs. Binding levels were increased in BMMs treated with SN‐rDPP4 and were abolished by STING knockdown ( n = 6 biological replicates). Data are shown as mean ± SD.

Article Snippet: Conversely, rDPP4 (Sino Biological, 10688‐H08H) administration involved once‐weekly intramedullary injections (0.2 μg per injection) for 4 weeks.

Techniques: Analysis, Western Blot, Activity Assay, Knockdown, Cell Culture, Luciferase, Transfection, Plasmid Preparation, Chromatin Immunoprecipitation, Binding Assay

Journal: Aging Cell

Article Title: Characterizing the SASP ‐Dependent Paracrine Spreading of Senescence Between Human Brain Cell Types

doi: 10.1111/acel.70673

Figure Lengend Snippet: Analysis of ligands and receptors in senescent and receiving cells. (A) Schematic depicting BulkSignalR pipeline which uses known ligand‐receptor interactions and affected downstream pathways to analyze their activation based on our bulk RNAseq data from DMSO and BrdU treated human cell lines (created with BioRender). (B) Venn diagram showing the number of receptors inferred from BulkSignalR to be activated across each of the five human cell types. Three receptors were identified in common between astrocytes (purple), endothelial cells (pink), and microglia (yellow) which were the cell types shown (Figure ) to be capable of receiving senescence signals and becoming SA β‐gal positive: CXCR7, KREMEN2, and GIPR. Only CXCR7 was expressed in the cell types capable of entering secondary senescence (astrocytes, endothelial cells, microglia) (Figure , Figure ). (C) TPM expression values of CXCR7 , its ligand CXCL12 , and DPP4 which cleaves and inactivates CXCL12 in DMSO (gray) and BrdU (red) treated cell lines ( n = 3 replicates). (D) Schematic of the four selected SASP inhibitors mechanisms of action: Bindarit is a CCL2 synthesis inhibitor which prevents p65 activation of the CCL2 gene at the promoter region, ISO‐1 is a MIF antagonist, ACT‐1004‐1239 is a CXCR7 antagonist, and Sitagliptin inhibits DPP4 preventing its action of cleaving and inactivating CXCL12 (created with BioRender). Data were analyzed by two‐way ANOVA with Tukey's multiple comparisons test (C). All graphs show mean with error bars depicting standard deviation (ns, p > 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: Treatment with DPP4 inhibitor Sitagliptin (MedChemExpress, Catalog No. HY‐13749) was used to eliminate DPP4‐dependent cleavage and inactivation of CXCL12, which is a ligand of the CXCR7 receptor.

Techniques: Activation Assay, RNA sequencing, Expressing, Standard Deviation

Journal: The Journal of Clinical Endocrinology and Metabolism

Article Title: HMGCR and Rosuvastatin Regulates GLP-1 Secretion and Expression—A Translational Study

doi: 10.1210/clinem/dgaf608

Figure Lengend Snippet: A to F, Oral glucose tolerance test (OGTT) performed after rosuvastatin (0.2 mg) treatment once daily for 27 days. A, Rosuvastatin-treated mice exhibit lower 60- and 90-minute plasma glucose levels, but B, area under the curve (AUC) was unaffected for glucose. C, Rosuvastatin-treated mice exhibit higher 5- and 60-minute plasma glucagon-like peptide 1 (GLP-1) levels, but D, AUC was unaffected for GLP-1. E, Rosuvastatin-treated mice had higher insulin secretion; AUC is presented in F. G, Long-term administration of rosuvastatin had no effect on the number of L cells in the jejunum. H to J, Long-term administration of rosuvastatin had no effect on intestinal messenger RNA expression of Gcg , Gip , Pyy , Pcsk1 , or Dpp4 in the H, duodenum; I, jejunum; or J, ileum. K, Long-term administration of rosuvastatin had no effect on β-cell mass. All parameters were assessed in 10 mice per group. Data are presented as box and whisker plots showing the minimum and maximum values (bottom and top error bars), the first (bottom of the box) and third (top of the box) quartiles and the median (middle of the box), or as a line graph showing the mean ± SEM. * P less than .05; ** P less than .01; and *** P less than .001 compared with vehicle-treated mice.

Article Snippet: RNA was reverse-transcribed using RevertAid First Strand complementary DNA synthesis kit (Thermo Scientific). qPCR for Dpp4 (Mm00494538_m1), Gip (Mm00433601_m1) Hmgcr (Mm01282499_m1), Proglucagon ( Gcg ; Mm01269055_m1), Pcsk1 (Mm00479023_m1), Pyy (Mm00520715_m1), and 2 housekeeping genes ( Hprt [Mm03024075_m1] and Tbp [Mm01277042_m1]) was performed using TaqMan Expression PCR Master Mix (Life Technologies) using the ABI Prism 7900 HT system (Applied Biosystems).

Techniques: Clinical Proteomics, RNA Expression, Whisker Assay