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Combined pharmacological inhibition of STING and NMN supplementation enhances osteogenic differentiation of BMSCs to mitigate age‐related bone loss. (a) Representative images of tetramethylrhodamine ethyl ester (TMRE) staining of mitochondrial membrane potential in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs (scale bar: 50 µm). (b) Representative images <t>of</t> <t>Calcein‐AM</t> (green) and MitoTracker (red) staining for mitochondrial permeability transition pore opening in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs (scale bar: 50 µm). (c) Seahorse analysis of OCR in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs, with quantitative analysis of basal and maximal respiration levels ( n = 6 biological replicates). (d) Representative images of Alizarin Red (red) and ALP (purple) staining for osteogenic differentiation activity in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs. (e) Representative immunoblots of RUN2 and BMP2 protein levels in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs ( n = 3 biological replicates). (f) Representative images of TMRE staining of mitochondrial membrane potential in BMSCs from 3‐ or 18‐month‐old AAV9‐BMM‐shNC‐treated or 18‐month‐old AAV9‐BMM‐sh Cd38 ‐treated mice (scale bar: 50 µm). (g) Representative images of Calcein‐AM (green) and MitoTracker (red) staining for mitochondrial permeability transition pore opening in BMSCs from 3‐ or 18‐month‐old AAV9‐BMM‐shNC‐treated or 18‐month‐old AAV9‐BMM‐sh Cd38 ‐treated mice (scale bar: 50 µm). (h) OCR analysis in BMSCs from AAV9‐BMM‐shNC‐treated 3 M‐BMSCs or AAV9‐BMM‐sh Cd38 ‐treated 3 M‐/18 M‐BMSCs, with quantitative analysis of basal and maximal respiration levels ( n = 6 biological replicates). (i) Representative images of Alizarin Red (red) and ALP (purple) staining for osteogenic differentiation activity in BMSCs from 3‐ or 18‐month‐old AAV9‐BMM‐shNC‐treated or 18‐month‐old AAV9‐BMM‐sh Cd38 ‐treated mice. (j) Representative immunoblots of RUNX2 and BMP2 protein levels in BMSCs from 3‐ or 18‐month‐old AAV9‐BMM‐shNC‐treated or 18‐month‐old AAV9‐BMM‐sh Cd38 ‐treated mice ( n = 3 biological replicates). (k, l) Representative micro‐CT images (k) and quantitative analysis of BV/TV, Tb. N, and Tb.Sp (l) from PBS‐treated, ALD‐treated, or H‐151 and NMN co‐treated mice ( n = 6 biological replicates). Data are shown as mean ± SD.
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Combined pharmacological inhibition of STING and NMN supplementation enhances osteogenic differentiation of BMSCs to mitigate age‐related bone loss. (a) Representative images of tetramethylrhodamine ethyl ester (TMRE) staining of mitochondrial membrane potential in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs (scale bar: 50 µm). (b) Representative images <t>of</t> <t>Calcein‐AM</t> (green) and MitoTracker (red) staining for mitochondrial permeability transition pore opening in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs (scale bar: 50 µm). (c) Seahorse analysis of OCR in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs, with quantitative analysis of basal and maximal respiration levels ( n = 6 biological replicates). (d) Representative images of Alizarin Red (red) and ALP (purple) staining for osteogenic differentiation activity in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs. (e) Representative immunoblots of RUN2 and BMP2 protein levels in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs ( n = 3 biological replicates). (f) Representative images of TMRE staining of mitochondrial membrane potential in BMSCs from 3‐ or 18‐month‐old AAV9‐BMM‐shNC‐treated or 18‐month‐old AAV9‐BMM‐sh Cd38 ‐treated mice (scale bar: 50 µm). (g) Representative images of Calcein‐AM (green) and MitoTracker (red) staining for mitochondrial permeability transition pore opening in BMSCs from 3‐ or 18‐month‐old AAV9‐BMM‐shNC‐treated or 18‐month‐old AAV9‐BMM‐sh Cd38 ‐treated mice (scale bar: 50 µm). (h) OCR analysis in BMSCs from AAV9‐BMM‐shNC‐treated 3 M‐BMSCs or AAV9‐BMM‐sh Cd38 ‐treated 3 M‐/18 M‐BMSCs, with quantitative analysis of basal and maximal respiration levels ( n = 6 biological replicates). (i) Representative images of Alizarin Red (red) and ALP (purple) staining for osteogenic differentiation activity in BMSCs from 3‐ or 18‐month‐old AAV9‐BMM‐shNC‐treated or 18‐month‐old AAV9‐BMM‐sh Cd38 ‐treated mice. (j) Representative immunoblots of RUNX2 and BMP2 protein levels in BMSCs from 3‐ or 18‐month‐old AAV9‐BMM‐shNC‐treated or 18‐month‐old AAV9‐BMM‐sh Cd38 ‐treated mice ( n = 3 biological replicates). (k, l) Representative micro‐CT images (k) and quantitative analysis of BV/TV, Tb. N, and Tb.Sp (l) from PBS‐treated, ALD‐treated, or H‐151 and NMN co‐treated mice ( n = 6 biological replicates). Data are shown as mean ± SD.
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Combined pharmacological inhibition of STING and NMN supplementation enhances osteogenic differentiation of BMSCs to mitigate age‐related bone loss. (a) Representative images of tetramethylrhodamine ethyl ester (TMRE) staining of mitochondrial membrane potential in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs (scale bar: 50 µm). (b) Representative images <t>of</t> <t>Calcein‐AM</t> (green) and MitoTracker (red) staining for mitochondrial permeability transition pore opening in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs (scale bar: 50 µm). (c) Seahorse analysis of OCR in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs, with quantitative analysis of basal and maximal respiration levels ( n = 6 biological replicates). (d) Representative images of Alizarin Red (red) and ALP (purple) staining for osteogenic differentiation activity in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs. (e) Representative immunoblots of RUN2 and BMP2 protein levels in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs ( n = 3 biological replicates). (f) Representative images of TMRE staining of mitochondrial membrane potential in BMSCs from 3‐ or 18‐month‐old AAV9‐BMM‐shNC‐treated or 18‐month‐old AAV9‐BMM‐sh Cd38 ‐treated mice (scale bar: 50 µm). (g) Representative images of Calcein‐AM (green) and MitoTracker (red) staining for mitochondrial permeability transition pore opening in BMSCs from 3‐ or 18‐month‐old AAV9‐BMM‐shNC‐treated or 18‐month‐old AAV9‐BMM‐sh Cd38 ‐treated mice (scale bar: 50 µm). (h) OCR analysis in BMSCs from AAV9‐BMM‐shNC‐treated 3 M‐BMSCs or AAV9‐BMM‐sh Cd38 ‐treated 3 M‐/18 M‐BMSCs, with quantitative analysis of basal and maximal respiration levels ( n = 6 biological replicates). (i) Representative images of Alizarin Red (red) and ALP (purple) staining for osteogenic differentiation activity in BMSCs from 3‐ or 18‐month‐old AAV9‐BMM‐shNC‐treated or 18‐month‐old AAV9‐BMM‐sh Cd38 ‐treated mice. (j) Representative immunoblots of RUNX2 and BMP2 protein levels in BMSCs from 3‐ or 18‐month‐old AAV9‐BMM‐shNC‐treated or 18‐month‐old AAV9‐BMM‐sh Cd38 ‐treated mice ( n = 3 biological replicates). (k, l) Representative micro‐CT images (k) and quantitative analysis of BV/TV, Tb. N, and Tb.Sp (l) from PBS‐treated, ALD‐treated, or H‐151 and NMN co‐treated mice ( n = 6 biological replicates). Data are shown as mean ± SD.
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Combined pharmacological inhibition of STING and NMN supplementation enhances osteogenic differentiation of BMSCs to mitigate age‐related bone loss. (a) Representative images of tetramethylrhodamine ethyl ester (TMRE) staining of mitochondrial membrane potential in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs (scale bar: 50 µm). (b) Representative images <t>of</t> <t>Calcein‐AM</t> (green) and MitoTracker (red) staining for mitochondrial permeability transition pore opening in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs (scale bar: 50 µm). (c) Seahorse analysis of OCR in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs, with quantitative analysis of basal and maximal respiration levels ( n = 6 biological replicates). (d) Representative images of Alizarin Red (red) and ALP (purple) staining for osteogenic differentiation activity in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs. (e) Representative immunoblots of RUN2 and BMP2 protein levels in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs ( n = 3 biological replicates). (f) Representative images of TMRE staining of mitochondrial membrane potential in BMSCs from 3‐ or 18‐month‐old AAV9‐BMM‐shNC‐treated or 18‐month‐old AAV9‐BMM‐sh Cd38 ‐treated mice (scale bar: 50 µm). (g) Representative images of Calcein‐AM (green) and MitoTracker (red) staining for mitochondrial permeability transition pore opening in BMSCs from 3‐ or 18‐month‐old AAV9‐BMM‐shNC‐treated or 18‐month‐old AAV9‐BMM‐sh Cd38 ‐treated mice (scale bar: 50 µm). (h) OCR analysis in BMSCs from AAV9‐BMM‐shNC‐treated 3 M‐BMSCs or AAV9‐BMM‐sh Cd38 ‐treated 3 M‐/18 M‐BMSCs, with quantitative analysis of basal and maximal respiration levels ( n = 6 biological replicates). (i) Representative images of Alizarin Red (red) and ALP (purple) staining for osteogenic differentiation activity in BMSCs from 3‐ or 18‐month‐old AAV9‐BMM‐shNC‐treated or 18‐month‐old AAV9‐BMM‐sh Cd38 ‐treated mice. (j) Representative immunoblots of RUNX2 and BMP2 protein levels in BMSCs from 3‐ or 18‐month‐old AAV9‐BMM‐shNC‐treated or 18‐month‐old AAV9‐BMM‐sh Cd38 ‐treated mice ( n = 3 biological replicates). (k, l) Representative micro‐CT images (k) and quantitative analysis of BV/TV, Tb. N, and Tb.Sp (l) from PBS‐treated, ALD‐treated, or H‐151 and NMN co‐treated mice ( n = 6 biological replicates). Data are shown as mean ± SD.
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Combined pharmacological inhibition of STING and NMN supplementation enhances osteogenic differentiation of BMSCs to mitigate age‐related bone loss. (a) Representative images of tetramethylrhodamine ethyl ester (TMRE) staining of mitochondrial membrane potential in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs (scale bar: 50 µm). (b) Representative images <t>of</t> <t>Calcein‐AM</t> (green) and MitoTracker (red) staining for mitochondrial permeability transition pore opening in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs (scale bar: 50 µm). (c) Seahorse analysis of OCR in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs, with quantitative analysis of basal and maximal respiration levels ( n = 6 biological replicates). (d) Representative images of Alizarin Red (red) and ALP (purple) staining for osteogenic differentiation activity in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs. (e) Representative immunoblots of RUN2 and BMP2 protein levels in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs ( n = 3 biological replicates). (f) Representative images of TMRE staining of mitochondrial membrane potential in BMSCs from 3‐ or 18‐month‐old AAV9‐BMM‐shNC‐treated or 18‐month‐old AAV9‐BMM‐sh Cd38 ‐treated mice (scale bar: 50 µm). (g) Representative images of Calcein‐AM (green) and MitoTracker (red) staining for mitochondrial permeability transition pore opening in BMSCs from 3‐ or 18‐month‐old AAV9‐BMM‐shNC‐treated or 18‐month‐old AAV9‐BMM‐sh Cd38 ‐treated mice (scale bar: 50 µm). (h) OCR analysis in BMSCs from AAV9‐BMM‐shNC‐treated 3 M‐BMSCs or AAV9‐BMM‐sh Cd38 ‐treated 3 M‐/18 M‐BMSCs, with quantitative analysis of basal and maximal respiration levels ( n = 6 biological replicates). (i) Representative images of Alizarin Red (red) and ALP (purple) staining for osteogenic differentiation activity in BMSCs from 3‐ or 18‐month‐old AAV9‐BMM‐shNC‐treated or 18‐month‐old AAV9‐BMM‐sh Cd38 ‐treated mice. (j) Representative immunoblots of RUNX2 and BMP2 protein levels in BMSCs from 3‐ or 18‐month‐old AAV9‐BMM‐shNC‐treated or 18‐month‐old AAV9‐BMM‐sh Cd38 ‐treated mice ( n = 3 biological replicates). (k, l) Representative micro‐CT images (k) and quantitative analysis of BV/TV, Tb. N, and Tb.Sp (l) from PBS‐treated, ALD‐treated, or H‐151 and NMN co‐treated mice ( n = 6 biological replicates). Data are shown as mean ± SD.
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Combined pharmacological inhibition of STING and NMN supplementation enhances osteogenic differentiation of BMSCs to mitigate age‐related bone loss. (a) Representative images of tetramethylrhodamine ethyl ester (TMRE) staining of mitochondrial membrane potential in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs (scale bar: 50 µm). (b) Representative images of Calcein‐AM (green) and MitoTracker (red) staining for mitochondrial permeability transition pore opening in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs (scale bar: 50 µm). (c) Seahorse analysis of OCR in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs, with quantitative analysis of basal and maximal respiration levels ( n = 6 biological replicates). (d) Representative images of Alizarin Red (red) and ALP (purple) staining for osteogenic differentiation activity in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs. (e) Representative immunoblots of RUN2 and BMP2 protein levels in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs ( n = 3 biological replicates). (f) Representative images of TMRE staining of mitochondrial membrane potential in BMSCs from 3‐ or 18‐month‐old AAV9‐BMM‐shNC‐treated or 18‐month‐old AAV9‐BMM‐sh Cd38 ‐treated mice (scale bar: 50 µm). (g) Representative images of Calcein‐AM (green) and MitoTracker (red) staining for mitochondrial permeability transition pore opening in BMSCs from 3‐ or 18‐month‐old AAV9‐BMM‐shNC‐treated or 18‐month‐old AAV9‐BMM‐sh Cd38 ‐treated mice (scale bar: 50 µm). (h) OCR analysis in BMSCs from AAV9‐BMM‐shNC‐treated 3 M‐BMSCs or AAV9‐BMM‐sh Cd38 ‐treated 3 M‐/18 M‐BMSCs, with quantitative analysis of basal and maximal respiration levels ( n = 6 biological replicates). (i) Representative images of Alizarin Red (red) and ALP (purple) staining for osteogenic differentiation activity in BMSCs from 3‐ or 18‐month‐old AAV9‐BMM‐shNC‐treated or 18‐month‐old AAV9‐BMM‐sh Cd38 ‐treated mice. (j) Representative immunoblots of RUNX2 and BMP2 protein levels in BMSCs from 3‐ or 18‐month‐old AAV9‐BMM‐shNC‐treated or 18‐month‐old AAV9‐BMM‐sh Cd38 ‐treated mice ( n = 3 biological replicates). (k, l) Representative micro‐CT images (k) and quantitative analysis of BV/TV, Tb. N, and Tb.Sp (l) from PBS‐treated, ALD‐treated, or H‐151 and NMN co‐treated mice ( n = 6 biological replicates). Data are shown as mean ± SD.

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: Combined pharmacological inhibition of STING and NMN supplementation enhances osteogenic differentiation of BMSCs to mitigate age‐related bone loss. (a) Representative images of tetramethylrhodamine ethyl ester (TMRE) staining of mitochondrial membrane potential in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs (scale bar: 50 µm). (b) Representative images of Calcein‐AM (green) and MitoTracker (red) staining for mitochondrial permeability transition pore opening in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs (scale bar: 50 µm). (c) Seahorse analysis of OCR in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs, with quantitative analysis of basal and maximal respiration levels ( n = 6 biological replicates). (d) Representative images of Alizarin Red (red) and ALP (purple) staining for osteogenic differentiation activity in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs. (e) Representative immunoblots of RUN2 and BMP2 protein levels in WT‐3 M‐/WT‐18 M‐BMSCs and Sting ΔMφ ‐18 M‐BMSCs ( n = 3 biological replicates). (f) Representative images of TMRE staining of mitochondrial membrane potential in BMSCs from 3‐ or 18‐month‐old AAV9‐BMM‐shNC‐treated or 18‐month‐old AAV9‐BMM‐sh Cd38 ‐treated mice (scale bar: 50 µm). (g) Representative images of Calcein‐AM (green) and MitoTracker (red) staining for mitochondrial permeability transition pore opening in BMSCs from 3‐ or 18‐month‐old AAV9‐BMM‐shNC‐treated or 18‐month‐old AAV9‐BMM‐sh Cd38 ‐treated mice (scale bar: 50 µm). (h) OCR analysis in BMSCs from AAV9‐BMM‐shNC‐treated 3 M‐BMSCs or AAV9‐BMM‐sh Cd38 ‐treated 3 M‐/18 M‐BMSCs, with quantitative analysis of basal and maximal respiration levels ( n = 6 biological replicates). (i) Representative images of Alizarin Red (red) and ALP (purple) staining for osteogenic differentiation activity in BMSCs from 3‐ or 18‐month‐old AAV9‐BMM‐shNC‐treated or 18‐month‐old AAV9‐BMM‐sh Cd38 ‐treated mice. (j) Representative immunoblots of RUNX2 and BMP2 protein levels in BMSCs from 3‐ or 18‐month‐old AAV9‐BMM‐shNC‐treated or 18‐month‐old AAV9‐BMM‐sh Cd38 ‐treated mice ( n = 3 biological replicates). (k, l) Representative micro‐CT images (k) and quantitative analysis of BV/TV, Tb. N, and Tb.Sp (l) from PBS‐treated, ALD‐treated, or H‐151 and NMN co‐treated mice ( n = 6 biological replicates). Data are shown as mean ± SD.

Article Snippet: For the mPTP opening assay, cells were first incubated with MitoTracker Red (Beyotime, C1032) at 37°C for 30 min to visualize mitochondria, then stained with calcein‐AM solution (Beyotime, C2009) at 37°C for 30 min.

Techniques: Inhibition, Staining, Membrane, Permeability, Analysis, Activity Assay, Western Blot, Micro-CT