Journal: Materials Today Bio

Article Title: Extracellular biogenic nanoscale mitochondria reprogram the wound microenvironment via ROS scavenging independent of cellular uptake

doi: 10.1016/j.mtbio.2026.103023

Figure Lengend Snippet: MSC-mt internalization promotes mitophagy activation under oxidative stress (A-B) Flow cytometric analysis of mitophagy in L929 cells co-cultured with fluorescently labeled MSC-mt under H 2 O 2 -induced oxidative stress. Mitophagy levels are shown for total cells as well as stratified mt transfer + and mt transfer − subpopulations, showing preferential mitophagy activation in mt transfer + cells. (C-D) Western blot analysis of mitophagy- and survival-related signaling proteins in flow-sorted mt transfer + and mt transfer − L929 cells following co-culture with fluorescently labeled MSC-mt under oxidative stress. Blots show phosphorylated PINK1 (S228), total PINK1, Parkin, total p62, phosphorylated p62 (S349 and S403), pAKT, OXPHOS components, and TOM20, highlighting enhanced PINK1–Parkin signaling and mitophagy-associated p62 processing in mt transfer + cells. (E) Flow cytometric assessment of mitophagy in total, mt transfer + , and mt transfer − populations following co-culture with PINK1-deficient MSC-derived mitochondria (siPINK1-mt) under oxidative stress, showing attenuated mitophagy activation compared with control MSC-mt. (F) Representative immunofluorescence images of L929 cells under control, H 2 O 2 , and H 2 O 2 + MSC-mt conditions, showing depolarized mitochondria (mitoPeDPP, green) and mitophagy signals (mitophagy, red), indicating increased mitophagic engagement under oxidative stress with MSC-mt transfer. Scale bar = 20 μm. (G–J) Flow cytometric analysis of depolarized mitochondria (mitoPeDPP) and mitophagy in L929 cells under H 2 O 2 stimulation with or without fluorescently labeled MSC-mt co-culture. (G) Representative flow cytometry plots. (H) Quantification of the proportions of mitoPeDPP + , mitophagy + , and double-positive cell populations. (I) Mean fluorescence intensity (MFI) of mitophagy signals, with stratification by mt transfer + and mt transfer − populations. (J) MFI of mitoPeDPP signals, with stratification by mt transfer + and mt transfer − populations. All experiments were independently repeated three times (n = 3) and representative images are shown. Data are presented as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: In selected experiments, mitophagy was analyzed in parallel with mitochondrial depolarization using a dual-staining strategy combining the Mitophagy Detection Kit with the mitochondrial depolarization probe mitoPeDPP (Dojindo, Cat# M466), allowing simultaneous evaluation of mitophagy activation and damaged mitochondria at the single-cell level.

Techniques: Activation Assay, Cell Culture, Labeling, Western Blot, Co-Culture Assay, Derivative Assay, Control, Immunofluorescence, Flow Cytometry, Fluorescence

Journal: Materials Today Bio

Article Title: Extracellular biogenic nanoscale mitochondria reprogram the wound microenvironment via ROS scavenging independent of cellular uptake

doi: 10.1016/j.mtbio.2026.103023

Figure Lengend Snippet: MSC-mt mitigate oxidative damage and improve mitochondrial function in H 2 O 2 -treated skin fibroblasts. (A–B) Representative flow cytometry plots and quantification of Annexin V/PI staining for apoptosis after H 2 O 2 stimulation, showing that MSC-mt significantly reduce fibroblast apoptosis, whereas rotenone-pretreated mitochondria (mt(R)) exhibit markedly attenuated protective effects. (C–D) Representative plots and quantification of mitoSOX staining for mitochondrial ROS after 180 min H 2 O 2 exposure, showing that MSC-mt suppress mitochondrial ROS accumulation, an effect largely lost following rotenone pretreatment. (E–F) Representative plots and quantification of TMRE staining for mitochondrial membrane potential (ΔΨm) after 180 min H 2 O 2 treatment, showing preservation of mitochondrial membrane potential by MSC-mt but not by mt(R). (G–H) Representative plots and quantification of SA-β-gal staining for cellular senescence after 180 min H 2 O 2 treatment, showing reduced senescence burden in MSC-mt–treated cells, with diminished efficacy observed in the mt(R) group. (I) Representative images and quantification of colony formation assays on day 8 post-treatment, indicating improved long-term proliferative capacity following MSC-mt treatment, an effect substantially weakened following rotenone pretreatment. (J) Heatmap of differentially expressed genes identified by a wound healing PCR array following 180 min of H 2 O 2 stimulation, showing that MSC-mt partially reverse H 2 O 2 -induced transcriptional alterations associated with stress response and repair pathways, an effect attenuated when using mt(R). (K) Representative images of mouse apoptosis protein array analysis after 180 min of H 2 O 2 treatment, showing that MSC-mt partially restore the expression of key anti-apoptotic and pro-survival signaling proteins suppressed by oxidative stress, including phosphorylated AKT (Ser473), BAD (Ser112), ERK (T202), and IκBα (S32), with reduced restoration observed in the mt(R) group. (L) Western blot analysis of phosphorylated AKT at Ser473 expression after 180 min of H 2 O 2 exposure, showing restoration of pro-survival signaling by MSC-mt, which is attenuated in the mt(R) group. All experiments were independently repeated three times (n = 3). Data are presented as mean ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ns, not significant.

Article Snippet: The mitochondrial membrane potential (ΔΨm) of isolated MSC-mt was assessed using a TMRE Mitochondrial Membrane Potential Assay Kit (Beyotime, Cat# C2001S).

Techniques: Flow Cytometry, Staining, Membrane, Preserving, Protein Array, Expressing, Western Blot

Journal: iScience

Article Title: CGF induces ROS-mediated metabolic reprogramming and mitochondrial dysfunction to suppress colorectal cancer progression

doi: 10.1016/j.isci.2026.115273

Figure Lengend Snippet: CGF’s effect on ABC transporter pathway, mitochondrial function, and ROS in CRC (A) Flow cytometry analysis of the effect of CGF on ROS levels in HCT116 and HT29 cells. Cells were stained with DCFH-DA, a ROS probe, and fluorescence intensity was measured. The lower panel shows the relative percentage of ROS levels in HCT116 and HT29 cells under different treatments. (B) TEM observation of mitochondrial morphology in HCT116 (top) and HT29 (bottom) cells treated with CGF (50 μM, 24h). Arrows indicate normal mitochondrial morphology (scale bar, 20 μM). (C) JC-1 staining was used to assess how CGF treatment affects the mitochondrial membrane potential in HCT116 and HT29 cells. The change in mitochondrial membrane potential is indicated by the red to green fluorescence ratio (scale bar, 20 μM). (D and E) Assessment of SOD (upper) and CAT (lower) enzyme activities in HCT116 and HT29 cells following CGF exposure. (F and G) RT-qPCR was used to analyze the relative expression levels of ABC transporter genes such as ABCA1 , ABCC2 , ABCB5 , and CFTR in HCT116 (F) and HT29 (G) cells exposed to varying concentrations of CGF. (H) Assays for ATP detection demonstrate the impact of CGF on ATP levels within HCT116 and HT29 cells, with four biological replicates. (I) Flow cytometry analysis of the effect of ATP on ROS levels in HCT116 and HT29 cells. The right panel shows the relative percentage of ROS levels in HCT116 and HT29 cells after ATP treatment. (A, D–I) Data presentation is in the form of mean ± SEM. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; ∗∗∗∗ p < 0.0001.

Article Snippet: Enhanced mitochondrial membrane potential assay kit with JC-1 , Beyotime , Cat #C2003S.

Techniques: Flow Cytometry, Staining, Fluorescence, Membrane, Quantitative RT-PCR, Expressing

Journal: Nucleic Acids Research

Article Title: Development and application of G4-Flame as a visual biosensor for G4-DNA

doi: 10.1093/nar/gkag179

Figure Lengend Snippet: G4-Flame visualizes G4 Structures in cell mitochondria. ( A ) Schematic diagram of Mito-G4-Flame cell-line construction. Green: MTS sequence, yellow: G4-Flame sequence, and blue: 3× flag tag sequence. Arrows of different colors in the plasmid represent the corresponding functional elements shown in the diagram. ( B, C ). ATP levels and mitochondrial membrane potential (JC-1) in 293T cells transiently transfected with Mito-G4-Flame, compared with empty vector controls. No significant differences were observed between the two groups after transfection. ( D – F ) Subcellular fractionation assay validating Mito-G4-Flame mitochondrial targeting. WB probed with: anti-Tomm70 (mitochondrial marker), anti-GAPDH (cytoplasmic marker), anti-Actin (cytoplasmic marker), and anti-Flag (detecting G4-Flame). Results confirm exclusive mitochondrial localization of Mito-G4-Flame in stably transfected (D) HEK293T, (E) PLC, and (F) SNU 449 cells. Fluorescence images ( G, I , and K ) and co-localization analysis ( H, J , and M ) of HEK293T, PLC, and SNU 449 cells expressing Mito-G4-Flame (ex 485/405 nm, em 520 nm) costained with anti-ATP5A1 (mitochondrial marker). Scale bars: 10 µm. Data information: (B, C) are mean ± SD, unpaired t -test; ns, not significant.

Article Snippet: Twenty-four hours after transfection, the mitochondrial membrane potential was measured using the Beyotime Enhanced Mitochondrial Membrane Potential Assay Kit with JC-1.

Techniques: Sequencing, FLAG-tag, Plasmid Preparation, Functional Assay, Membrane, Transfection, Fractionation, Marker, Stable Transfection, Fluorescence, Expressing

Journal: Nucleic Acids Research

Article Title: Development and application of G4-Flame as a visual biosensor for G4-DNA

doi: 10.1093/nar/gkag179

Figure Lengend Snippet: G4-Flame reveals mitochondrial G4-DNA suppresses mtDNA expression. Fluorescence images ( A ) and quantitative analysis ( B ) of SNU449 cells stably expressing Mito-G4-Flame. Cells were treated with 100 µM PDS for 2, 4, 8, and 10 h at 37 °C, showing a continuous decrease in R485/405 ratios over time. Scale bars: 10 µm. ( C ) PCR analysis of mitochondrial-encoded gene expression in SNU449 cells following PDS treatment across the indicated time points. Heatmap representation shows a time-dependent decrease in mRNA levels with increasing duration of PDS treatment. ( D ) WB analysis showing overexpression of mito-DHX36 in SNU449 cells stably expressing the construct. A strong DHX36 signal is observed, indicating robust expression in the mito-DHX36 stable cell line. Fluorescence images ( E ), colocalization analysis ( F ), and quantitative analysis ( G ) of SNU449 cells stably expressing mito-DHX36. DHX36 shows clear colocalization with mitochondria, and overexpression of mito-DHX36 leads to increased fluorescence intensity. Scale bars: 10 µm. Fluorescence images ( H ) and quantitative analysis ( I ) of SNU449 cells stably expressing Mito-G4-Flame with DHX36 overexpression. Overexpression of DHX36 in the stable cell line leads to an increase in R485/405 ratios. Scale bars: 10 µm. ( J ) qPCR analysis of mitochondrial-encoded gene expression in SNU449 cells stably expressing mito-DHX36. Overexpression of mito-DHX36 in the stable cell line led to increased expression of most mitochondrial-encoded genes. Data information: (J) are mean ± SD, (B) one-way ANOVA with Dunnett’s multiple comparisons test, (G and I) unpaired t- test, (J) two-way ANOVA with Dunnett’s multiple comparisons test; ns, not significant; * P < .05, ** P < .01, *** P < .001, **** P < .0001.

Article Snippet: Twenty-four hours after transfection, the mitochondrial membrane potential was measured using the Beyotime Enhanced Mitochondrial Membrane Potential Assay Kit with JC-1.

Techniques: Expressing, Fluorescence, Stable Transfection, Gene Expression, Over Expression, Construct

Journal: Materials Today Bio

Article Title: Orchestrating diabetic wound repair via mitochondria-targeted delivery of dihydromyricetin with tailored ADSC-derived biohybrid nanovesicles

doi: 10.1016/j.mtbio.2026.102934

Figure Lengend Snippet: Schematic illustration of synthesis and mechanism of biohybrid DHM@mtABV. (A) Fabrication process of DHM@mtLipo, ANV, and the biohybrid DHM@mtABV. (B) Cellular internalization, mitochondrial-targeted delivery and downstream regulatory pathways of DHM@mtABV. (C) Protection of cellular functions and mitochondrial homeostasis of DHM@mtABV in diabetic wound.

Article Snippet: Mitochondrial membrane potential was quantified with an enhanced JC-1 kit (Beyotime, China).

Techniques:

Journal: Materials Today Bio

Article Title: Orchestrating diabetic wound repair via mitochondria-targeted delivery of dihydromyricetin with tailored ADSC-derived biohybrid nanovesicles

doi: 10.1016/j.mtbio.2026.102934

Figure Lengend Snippet: Sustained oxidative stress and mitochondrial dysfunction impeded diabetic wound healing. (A) Representative images, monitoring illustration and corresponding statistics of wound healing process of control and diabetic mice. ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001, n = 5. (B) The representative HE staining of full-thickness wound. Abbreviation: ep, epithelium; et, proliferating epithelial tongues; de, dermis; sm, smooth muscle; gt, granulation tissue. The black, blue and white dashed line indicated the leading edge of et, gt and fibrin, respectively. The arrow highlights the advancing epithelium border. Scale bar: 1 mm. (C-D) The Masson staining and CD31 immunohistochemistry results of wound tissue. Scale bar: 100 μm. (E) DHE staining results. Scale bar: 200 μm. (F) Pathway synergy analysis of normal and diabetic wound. The subfigures display pathway synergy change plot, pathway activity correlation scatter plot, pathway activity boxplot and pathway correlation heatmap, respectively. Abbreviation: NRF2, NRF2 signaling; Antioxid, antioxidant defense; OxStress, oxidative stress; MitoMetab, mitochondrial metabolism; MitoDysfunc, mitochondrial dysfunction. (G) The Western blotting results of wound tissue protein, and corresponding differential analysis and correlation analysis (n = 5). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Mitochondrial membrane potential was quantified with an enhanced JC-1 kit (Beyotime, China).

Techniques: Control, Staining, Immunohistochemistry, Activity Assay, Western Blot

Journal: Materials Today Bio

Article Title: Orchestrating diabetic wound repair via mitochondria-targeted delivery of dihydromyricetin with tailored ADSC-derived biohybrid nanovesicles

doi: 10.1016/j.mtbio.2026.102934

Figure Lengend Snippet: Assessment of mitochondrial homeostasis in HUVEC. (A, B) Analysis of mitochondrial membrane potential using JC-1 staining. The representative results of mitochondrial JC-1 staining and corresponding quantitative analysis of the JC-1 aggregate/monomer ratio. Scale bar: 20 μm. (C) Evaluation of relative cellular ATP level. (D–F)​ Evaluation of mitochondrial stress. (D) The representative results of intracellular calcium levels detected by Rhod-2/AM staining, (E) total intracellular ROS levels measured by DCFH-DA staining, and (F) mitochondrial ROS assessed by MitoSOX Red staining. Scale bar: 30 μm. (G) The representative results of mPTP activity. The calcein staining after incubated with CoCl 2 (green) indicated intact mitochondria with closed mPTP pores. The treatment of cyclosporin A (CsA) inhibited the open of mPTP pore, which served as rescued group. Scale bar: 20 μm. (H) The corresponding statistical analysis (n = 3). ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: Mitochondrial membrane potential was quantified with an enhanced JC-1 kit (Beyotime, China).

Techniques: Membrane, Staining, Activity Assay, Incubation

Journal: Materials Today Bio

Article Title: Orchestrating diabetic wound repair via mitochondria-targeted delivery of dihydromyricetin with tailored ADSC-derived biohybrid nanovesicles

doi: 10.1016/j.mtbio.2026.102934

Figure Lengend Snippet: Oxidative damage is the key mechanism in delayed diabetic wound healing. (A) Single-cell RNA sequencing analysis of endothelial cells​ from cutaneous wounds. The panels (from left to right) show the UMAP visualization of pseudotime distribution, grouping, density distribution, and comparative pseudotime analysis across the four status groups (Non-diabetic, Non-DFU Diabetic, DFU-healer, and DFU-nonhealer). (B) Assessment of redox homeostasis pathway activity in endothelial cells. The boxplots show the separated and integrated activity scores of antioxidant metabolism and oxidative damage pathways scores, respectively. (C) Dynamic changes in antioxidant metabolism and oxidative damage pathway activities along the pseudotime trajectory within endothelial cells in different groups. (D) Correlation analysis between oxidative damage and mitochondrial function scores in endothelial cells, while the scatter plot includes a regression line indicating the overall trend. (E) The dose-response curve of ROS-scavenging capacity of DHM in a cell-free system under H 2 O 2 treatment, along with the fitted curve and corresponding photograph. (F) The quantitative analysis of ROS-scavenging capacity of ANV, DHM@mtLipo and DHM@mtABV in vitro in cell-free system. (G) The cytoprotective effect of concentration-gradient DHM in HUVEC with or without H 2 O 2 treatment measured with cell viability by CCK8 assay.

Article Snippet: Mitochondrial membrane potential was quantified with an enhanced JC-1 kit (Beyotime, China).

Techniques: Single Cell, RNA Sequencing, Activity Assay, In Vitro, Concentration Assay, CCK-8 Assay

Journal: Materials Today Bio

Article Title: Orchestrating diabetic wound repair via mitochondria-targeted delivery of dihydromyricetin with tailored ADSC-derived biohybrid nanovesicles

doi: 10.1016/j.mtbio.2026.102934

Figure Lengend Snippet: DHM@mtABV synergistically mitigates cellular oxidative stress through enhanced delivery efficiency and coordinated activation of antioxidant and proliferative pathways. (A) The Western blotting results reveal the effects of NRF2/HO-1 signaling activation of free DHM with gradient concentration. (B) Evaluation of intracellular ROS levels via DCFH-DA fluorescence staining in HUVECs. Scale bar: 20 μm. (C) Quantitative analysis of the cellular internalization efficiency of different DHM formulations (free DHM, DHM@mtLipo, and DHM@mtABV) measured by HPLC at 30 min post-treatment. (D)​ Assessment of intracellular and mitochondrial antioxidant capacity by measuring CAT, MDA, total SOD and mitochondrial SOD activity. (E)​ Western blot analysis and corresponding quantification of NRF2 expression and phosphorylation of AKT and ERK1/2 (n = 3).(F)​ Schematic diagram depicting the integrated mechanism in accelerating diabetic wound healing. ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001.

Article Snippet: Mitochondrial membrane potential was quantified with an enhanced JC-1 kit (Beyotime, China).

Techniques: Activation Assay, Western Blot, Concentration Assay, Fluorescence, Staining, Activity Assay, Expressing, Phospho-proteomics

Journal: Materials Today Bio

Article Title: Orchestrating diabetic wound repair via mitochondria-targeted delivery of dihydromyricetin with tailored ADSC-derived biohybrid nanovesicles

doi: 10.1016/j.mtbio.2026.102934

Figure Lengend Snippet: DHM@mtABV promoted proliferation, antioxidant, angiogenesis and mitochondrial health to accelerate diabetic wound repair. The representative results of (A) Ki67, PCNA, (B) TNF-α, DHE, (C) CD31 and α-SMA, and (D) NRF2, HO-1 immunofluorescence and SOD2 immunohistochemistry staining of wound sections (n = 3). The white arrow in (C) pointed the representative pattern of vessel. (E) The quantitative analysis of the results. (F) Schematic diagram illustrating the proposed mechanism of DHM@mtABV in promoting diabetic wound healing. Scale bar: 100 μm ∗ P < 0.05, ∗∗ P < 0.01, ∗∗∗ P < 0.001, ∗∗∗∗ P < 0.0001.

Article Snippet: Mitochondrial membrane potential was quantified with an enhanced JC-1 kit (Beyotime, China).

Techniques: Immunofluorescence, Immunohistochemistry, Staining

Journal: Materials Today Bio

Article Title: A versatile nanoplatform for enhancing the therapeutic efficacy against low-immunogenic TNBC by inducing immunogenic cell death and MHC-I upregulation

doi: 10.1016/j.mtbio.2026.102927

Figure Lengend Snippet: (a) Cell viability of 4T1 cells treated with PH and 3PNH, under light irradiation or in the dark. (“L” denotes light irradiation with a 660 nm laser at a power density of 0.5 W/cm 2 for 5 min) (n = 4). (b) Cell viability of 4T1 cells assessed by staining with Calcein-AM/PI under different treatment conditions. (c) Detection of apoptosis in 4T1 cells by Annexin V-FITC/PI dual staining, analyzed via flow cytometry under different treatment conditions(n = 3). (d) Visualization of mitochondrial membrane potential in 4T1 cells by TMRE staining under different treatment conditions. (e) Intracellular ATP levels in 4T1 cells under different treatment conditions (n = 3). Immunofluorescence staining was used to evaluate (f) the expression of CRT in 4T1 cells and (g) the release of HMGB-1 in the nuclei of 4T1 cells under different treatment conditions. Data are presented as mean ± SD. Statistical significance was calculated via one-way ANOVA with Tukey’ post hoc test. (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001).

Article Snippet: Calcein-AM/PI (cell viability and cytotoxicity detection kit), CCK-8 kit, Hoechst 33342, mitochondrial membrane potential detection kit (TMRE), and Annexin V-FITC apoptosis detection kit were all purchased from Beyotime Biotechnology Co., Ltd.

Techniques: Irradiation, Staining, Flow Cytometry, Membrane, Immunofluorescence, Expressing