Journal: Research

Article Title: Effect of Microcurrent Stimulation on Cartilage Regeneration in Engineered Chondrocyte Spheroids

doi: 10.34133/research.1462

Figure Lengend Snippet: Effects of microcurrent stimulation on chondrocyte proliferation in a 2-dimensional (2D) in vitro culture system. (A) Schematic illustration of chondrocytes cultured in 6-well plates under microcurrent stimulation. (B) Relative DNA content of chondrocytes in the 10-, 25-, 50-, 100-, and 200-μA groups compared with that of the control (Ctrl, 0 μA) group at day 9. (C) Relative optical density (OD) values of chondrocytes in the Ctrl and 10-, 25-, 50-, 100-, and 200-μA groups at day 5, as determined by the CCK-8 assay. (D) Representative Live/Dead staining images of chondrocytes in the Ctrl and 10-, 25-, 50-, 100-, and 200-μA groups from days 1 to 9. (E) Quantitative analysis of cell viability in the Ctrl and 10-, 25-, 50-, 100-, and 200-μA groups at day 9. (F) Representative 5-ethynyl-2′-deoxyuridine (EdU) staining images and corresponding quantification of the EdU-positive rate of chondrocytes in the Ctrl and 10-, 25-, 50-, 100-, and 200-μA groups at day 9. (G) Representative Ki67 staining images and corresponding quantification of the Ki67-positive rate of chondrocytes in the Ctrl and 10-, 25-, 50-, 100-, and 200-μA groups at day 9. Data are presented as mean ± SD ( n = 5). ns, not significant; *** P < 0.001; **** P < 0.0001.

Article Snippet: Samples were washed with PBS and incubated in working solution containing 2 μM calcein-AM and 4 μM propidium iodide using a calcein-AM/propidium iodide Live/Dead staining kit (Beyotime Biotechnology, Shanghai, China) for 20 min at 37 °C in the dark.

Techniques: In Vitro, Cell Culture, Control, CCK-8 Assay, Staining, Analysis

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

Journal: Advanced Science

Article Title: Targeting Nupr1‐Mediated p53 Mitochondrial Sequestration Alleviates Aortic Dissection by Restoring the Gadd45b‐Dependent Survival Program

doi: 10.1002/advs.78143

Figure Lengend Snippet: Genetic and Pharmacological Inhibition of Nupr1 Attenuates Ang II Induced Mitochondrial Apoptosis in VSMCs. (A) Cell ability was evaluated by CCK8 assay ( n = 8 per group). (B) Lactate production of indicated groups ( n = 6 per group). (C) Representative images of TEM in VSMCs from the indicated groups. (D) Quantification of mitochondrial size in VSMCs from indicated groups ( n = 6 per group). (E) Quantification of circularity index in VSMC from indicated groups ( n = 6 per group). (F) Real‐time monitoring of oxygen consumption rate (OCR) in indicated groups detected by O2k. (G–I) Quantification of ATP‐coupled respiration, mitochondrial basal respiration, and maximal respiration from indicated groups ( n = 6 per group). (J) Representative images of calcein and Annexin V fluorescence results showing mPTP opening and apoptosis in indicated groups. Stronger green fluorescence represents a lower degree of mPTP opening, while diminished fluorescence indicates increased mPTP opening. Stronger red fluorescence represents a higher apoptosis rate. (K, L) Quantification of fluorescence intensity, including Annexin V and Calcein ( n = 6 per group). (M) Flow cytometry analysis of Annexin and PI staining of apoptosis from indicated groups. (N) Quantification of early apoptosis, late apoptosis, and total apoptosis by flow cytometry ( n = 3 per group). (O) Quantification of maximal respiration from indicated groups ( n = 6 per group). (P) Real‐time monitoring of OCR in indicated groups detected by O2k. (Q, R) Quantification of ATP‐coupled respiration and mitochondrial basal respiration from indicated groups ( n = 6 per group). (S) Flow cytometry analysis of Annexin and PI staining of apoptosis from indicated groups. (T) Quantification of early apoptosis, late apoptosis, and total apoptosis by flow cytometry ( n = 3 per group).

Article Snippet: According to mPTP test kit instructions (C2009S, Beyotime), 200 μL of Calcein AM staining solution and fluorescence quenching working solution were added to each well after the respective cell treatments.

Techniques: Inhibition, CCK-8 Assay, Real-time Monitoring, Fluorescence, Flow Cytometry, Analysis, Staining

Journal: Advanced Science

Article Title: Targeting Nupr1‐Mediated p53 Mitochondrial Sequestration Alleviates Aortic Dissection by Restoring the Gadd45b‐Dependent Survival Program

doi: 10.1002/advs.78143

Figure Lengend Snippet: Gadd45b is Indispensable for the Protective Effects of Nupr1 Deficiency Against Mitochondrial Apoptosis. (A) Enrichment of KEGG pathways. (B) GSEA analysis in VSMCs treated with Ang II stimulation and Nupr1 deficiency. (C) Chordal diagram of GO pathways related with cell fate and the contributing DEPs. (D) Stem plot of DEPs in the apoptosis pathway from the indicated groups. (E) RT‐qPCR analysis of mRNA levels of candidate genes in VSMCs. Data were presented as relative fold change to controls ( n = 6 per group). (F) Expressions of Gadd45b in VSMCs measured by Western blot and its quantification from indicated groups measured by Western blot ( n = 5 per group). (G, H) Representative images of ROS staining in indicated groups. Semi‐quantitative analysis of mean fluorescence intensity in indicated groups ( n = 6 per group). (I) Representative images of calcein and Annexin V fluorescence results showing mPTP opening in indicated groups. (J) Quantification of fluorescence intensity, including Annexin V and mPTP ( n = 3 per group). (K) Co‐immunoprecipitation and Western blot assays of Myc‐Nupr1 and Gadd45b. (L) Co‐immunoprecipitation assays of Myc‐Gadd45b and Nupr1.

Article Snippet: According to mPTP test kit instructions (C2009S, Beyotime), 200 μL of Calcein AM staining solution and fluorescence quenching working solution were added to each well after the respective cell treatments.

Techniques: Analysis, Quantitative RT-PCR, Western Blot, Staining, Fluorescence, Co-Immunoprecipitation Assay, Significance Assay