Journal: Redox Biology

Article Title: Extracellular vesicle-mediated delivery of mitochondrial circRNA MTCO2 protects against cerebral ischemia by modulating mPTP-dependent ferroptosis ☆

doi: 10.1016/j.redox.2025.103806

Figure Lengend Snippet: Dual-targeted EV platform enables specific delivery of circMTCO2 to neuronal mitochondria. ( A ) Transmission electron microscopy images of EV Ctrl , RVG-EV RNA , RVG-EV mt-RNA . Scale bars, 100 nm. ( B ) Nanoparticle size distribution of indicated EVs. ( C ) Western blot of EV markers (Lamp2b, CD63, Tsg101) and Golgi marker (GM130) from cells and EVs. ( D ) Absolute quantification of circMTCO2 copies per EV by qPCR. ( E ) Relative expression of circMTCO2 in mitochondrial fractions of N2a cells treated with indicated EVs for 24 h. ( F ) Confocal fluorescence microscopy images of N2a cells treated with RVG-EV RNA-GFP and RVG-EV mt-RNA-GFP . Mitochondria stained with MitoTracker, and nuclei with DAPI. Notable colocalization was observed in the RVG-EV mt-RNA-GFP group. Data are presented as mean ± SEM (n = 3 per group). Statistical analysis was performed using unpaired two-tailed Student's t-test. ∗∗∗ P < 0.001; ns, not significant.

Article Snippet: Mitochondria in N2A cells were stained with 400 nM MitoTracker Deep Red (Thermo Fisher Scientific, USA) for 1 h, followed by washing with PBS.

Techniques: Transmission Assay, Electron Microscopy, Western Blot, Marker, Quantitative Proteomics, Expressing, Fluorescence, Microscopy, Staining, Two Tailed Test

Journal: Redox Biology

Article Title: Extracellular vesicle-mediated delivery of mitochondrial circRNA MTCO2 protects against cerebral ischemia by modulating mPTP-dependent ferroptosis ☆

doi: 10.1016/j.redox.2025.103806

Figure Lengend Snippet: circMTCO2 binds ANT1 to inhibit mPTP opening and preserve mitochondrial integrity. ( A ) LC-MS/MS identification of the representative peptide sequence of ANT1 pulled down by biotinylated circMTCO2 probe in N2a cells. ( B ) Western blot validation of ANT1 pulled down by biotinylated circMTCO2 probe, with GAPDH and VDAC1 served as cytoplasmic and mitochondrial controls. A biotin-labeled negative control (NC) probe was included to assess non-specific binding. ( C ) RNA immunoprecipitation (RIP) using an antibody against ANT1, followed by qRT-PCR for circMTCO2, showing enrichment of circMTCO2 in ANT1 immunoprecipitates. ( D ) Confocal fluorescence microscopy images showing colocalization of circMTCO2 FISH, ANT1 and Mitotracker in mitochondria. ( E ) Western blot and ( F ) RIP confirming enhanced ANT1-circMTCO2 binding after circMTCO2 overexpression in OGD N2a cells. ( G ) In vitro RNA-protein pulldown using truncated circMTCO2 probes reveals the 36–120 nt region as essential for ANT1 binding. ( H ) RIP assay showing significantly decreased interaction of ANT1 with mutant circMTCO2 compared to full-length circMTCO2. ( I ) Calcein-CoCl 2 quenching assay showing loss of mPTP-closing protection with mutant circMTCO2 (RVG-EV mt-mut RNA ). Scale bar, 5 μm. Data are presented as mean ± SEM (n = 3 per group). Statistical analysis was performed using unpaired two-tailed Student's t-test. ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001; ns, not significant.

Article Snippet: Mitochondria in N2A cells were stained with 400 nM MitoTracker Deep Red (Thermo Fisher Scientific, USA) for 1 h, followed by washing with PBS.

Techniques: Liquid Chromatography with Mass Spectroscopy, Sequencing, Western Blot, Biomarker Discovery, Labeling, Negative Control, Binding Assay, RNA Immunoprecipitation, Quantitative RT-PCR, Fluorescence, Microscopy, Over Expression, In Vitro, Mutagenesis, Two Tailed Test

Journal: Materials Today Bio

Article Title: ATG7-driven mitophagy in BMSC@CS hydrogel reprograms metabolism to boost bone regeneration

doi: 10.1016/j.mtbio.2025.102483

Figure Lengend Snippet: The effects of ATG7 overexpression in MSCs on mitochondrial function and energy metabolism. (A) Schematic illustration of the effects of ATG7 overexpression on mitochondrial function and energy metabolism in MSCs. (B) The mRNA levels of GLUT-3, GLUT-4, Ndufs1, Ndufs5, Ndufs6, Cyc1, CYTB and Uqcrfs1 were detected by RT‒qPCR in MSCs overexpressing ATG7. (C) ROS staining and (D) ROS FACs analysis of MSCs overexpressing ATG7. Scale bar = 75 μm. (E) TMRM staining and (F) TMRM FACs analysis of MSCs overexpressing ATG7. Scale bar = 75 μm. (G) lactate levels in the cell culture supernatant and (H) intracellular ATP levels were measured in MSCs with overexpression of ATG7.

Article Snippet: Intracellular ROS levels were assessed by incubating cells with 5 μM H 2 DCFDA (MCE, HY-D0940) in PBS for 30 min at 37 °C in the dark, followed by mounting with DAPI-containing medium (Solarbio, S2110) or staining with 1 mM mitochondrial tracker (MCE, HY-D1783) for 30 min ΔΨm was evaluated using 10 μM TMRM (MCE, HY-D0984A) under the same conditions.

Techniques: Over Expression, Staining, Cell Culture

Journal: Materials Today Bio

Article Title: ATG7-driven mitophagy in BMSC@CS hydrogel reprograms metabolism to boost bone regeneration

doi: 10.1016/j.mtbio.2025.102483

Figure Lengend Snippet: ATG7 Regulates Osteogenic Function via Mitochondrial Autophagy Activation in MSCs. (A) Western blot analysis of P62, Pink1, Parkin, and LC3 Ⅱ/Ⅰ protein expression in MSCs overexpressing Atg7. (B) Microscopy images and quantification of showing autophagy in BMSCs after overexpression ATG7. Mitochondria are stained green with Mitotracker, and lysosomes are stained red with Lysotracker. Yellow arrows indicate regions of mitochondrial and lysosomal co-localization. Scale bars: 25 μm. (C) Western blot analysis and quantification of P62, Pink1, Parkin, and LC3 Ⅱ/Ⅰ protein expression in Mdivi-1 (100 μM, 48 h) pretreated MSCs overexpressing ATG7. (D) RT‒qPCR analysis of osteogenic marker genes (ALP, OPN, OCN, and DMP-1) in ATG7-overexpressing MSCs pretreated with Mdivi-1 (100 μM) for 48 h. (E) ALP staining and quantitative analysis of osteoblast colony formation in Mdivi-1-pretreated MSCs overexpressing ATG7. Scale bar = 250 μm. (F) Western blot analysis and (G) quantification of AKT, p-AKT, PI3K, and p-PI3K protein expression in Mdivi-1 pretreated MSCs overexpressing ATG7. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Intracellular ROS levels were assessed by incubating cells with 5 μM H 2 DCFDA (MCE, HY-D0940) in PBS for 30 min at 37 °C in the dark, followed by mounting with DAPI-containing medium (Solarbio, S2110) or staining with 1 mM mitochondrial tracker (MCE, HY-D1783) for 30 min ΔΨm was evaluated using 10 μM TMRM (MCE, HY-D0984A) under the same conditions.

Techniques: Activation Assay, Western Blot, Expressing, Microscopy, Over Expression, Staining, Marker