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mitosox fluorescent probe  (MedChemExpress)


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    MedChemExpress mitosox fluorescent probe
    NAC alleviates ARG2-induced mitophagy suppression and cellular senescence in osteoblasts. MC3T3-E1 cells were transduced with pLJM1-EGFP control vector (Con) or pLJM1-HA-ARG2 (CMV-ARG2) to induce ARG2 overexpression. (A) Mitochondrial superoxide levels were assessed by <t>MitoSOX</t> staining (1 μM). (B) Mitochondrial membrane potential (Δψm) was evaluated by JC-1 staining. MC3T3-E1 cells were transduced with pLKO.1-control (Con) or pLKO.1-ARG2 shRNA (sh-ARG2) lentivirus for 48 h, followed by treatment with H₂O₂ (200 μM) for 24 h. (C) Mitochondrial superoxide levels were determined by MitoSOX staining (1 μM). (D) Δψm was assessed by JC-1 staining. For rescue experiments, MC3T3-E1 cells transduced with pLJM1-EGFP (Con) or pLJM1-HA-ARG2 (CMV-ARG2) were treated with N-acetylcysteine (NAC, 500 μM) for 24 h. (E) Mitochondrial superoxide levels were evaluated by MitoSOX staining (1 μM). (F) Δψm was assessed by JC-1 staining. (G) qRT-PCR analysis of PINK1 and Parkin mRNA expression. (H) Immunoblot analysis of p53, p21, p62, and LC3 protein expression. Quantitative analyses of protein levels are shown in the bar graphs on the right. (I) Senescence-associated β-galactosidase (SA-β-gal) staining. Bar graphs represent the quantification of SA-β-gal-positive cells. Statistical significance was determined by one-way ANOVA. Data are presented as mean ± SEM (n = 4). Scale bar = 100 μm. *p < 0.05, **p < 0.01, ***p < 0.001 between the indicated groups.
    Mitosox Fluorescent Probe, supplied by MedChemExpress, used in various techniques. Bioz Stars score: 99/100, based on 491 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/mitosox+red+fluorescent+probe/MitoSOX+Red/pmc13684819-133-10-13
    Average 99 stars, based on 491 article reviews
    mitosox fluorescent probe - by Bioz Stars, 2026-11
    99/100 stars

    Images

    1) Product Images from "ARG2 drives osteoblast senescence by repressing mitophagy via the IκB–NF-κB–TXN2 signaling axis: implications for age-associated osteoporosis"

    Article Title: ARG2 drives osteoblast senescence by repressing mitophagy via the IκB–NF-κB–TXN2 signaling axis: implications for age-associated osteoporosis

    Journal: Theranostics

    doi: 10.7150/thno.129818

    NAC alleviates ARG2-induced mitophagy suppression and cellular senescence in osteoblasts. MC3T3-E1 cells were transduced with pLJM1-EGFP control vector (Con) or pLJM1-HA-ARG2 (CMV-ARG2) to induce ARG2 overexpression. (A) Mitochondrial superoxide levels were assessed by MitoSOX staining (1 μM). (B) Mitochondrial membrane potential (Δψm) was evaluated by JC-1 staining. MC3T3-E1 cells were transduced with pLKO.1-control (Con) or pLKO.1-ARG2 shRNA (sh-ARG2) lentivirus for 48 h, followed by treatment with H₂O₂ (200 μM) for 24 h. (C) Mitochondrial superoxide levels were determined by MitoSOX staining (1 μM). (D) Δψm was assessed by JC-1 staining. For rescue experiments, MC3T3-E1 cells transduced with pLJM1-EGFP (Con) or pLJM1-HA-ARG2 (CMV-ARG2) were treated with N-acetylcysteine (NAC, 500 μM) for 24 h. (E) Mitochondrial superoxide levels were evaluated by MitoSOX staining (1 μM). (F) Δψm was assessed by JC-1 staining. (G) qRT-PCR analysis of PINK1 and Parkin mRNA expression. (H) Immunoblot analysis of p53, p21, p62, and LC3 protein expression. Quantitative analyses of protein levels are shown in the bar graphs on the right. (I) Senescence-associated β-galactosidase (SA-β-gal) staining. Bar graphs represent the quantification of SA-β-gal-positive cells. Statistical significance was determined by one-way ANOVA. Data are presented as mean ± SEM (n = 4). Scale bar = 100 μm. *p < 0.05, **p < 0.01, ***p < 0.001 between the indicated groups.
    Figure Legend Snippet: NAC alleviates ARG2-induced mitophagy suppression and cellular senescence in osteoblasts. MC3T3-E1 cells were transduced with pLJM1-EGFP control vector (Con) or pLJM1-HA-ARG2 (CMV-ARG2) to induce ARG2 overexpression. (A) Mitochondrial superoxide levels were assessed by MitoSOX staining (1 μM). (B) Mitochondrial membrane potential (Δψm) was evaluated by JC-1 staining. MC3T3-E1 cells were transduced with pLKO.1-control (Con) or pLKO.1-ARG2 shRNA (sh-ARG2) lentivirus for 48 h, followed by treatment with H₂O₂ (200 μM) for 24 h. (C) Mitochondrial superoxide levels were determined by MitoSOX staining (1 μM). (D) Δψm was assessed by JC-1 staining. For rescue experiments, MC3T3-E1 cells transduced with pLJM1-EGFP (Con) or pLJM1-HA-ARG2 (CMV-ARG2) were treated with N-acetylcysteine (NAC, 500 μM) for 24 h. (E) Mitochondrial superoxide levels were evaluated by MitoSOX staining (1 μM). (F) Δψm was assessed by JC-1 staining. (G) qRT-PCR analysis of PINK1 and Parkin mRNA expression. (H) Immunoblot analysis of p53, p21, p62, and LC3 protein expression. Quantitative analyses of protein levels are shown in the bar graphs on the right. (I) Senescence-associated β-galactosidase (SA-β-gal) staining. Bar graphs represent the quantification of SA-β-gal-positive cells. Statistical significance was determined by one-way ANOVA. Data are presented as mean ± SEM (n = 4). Scale bar = 100 μm. *p < 0.05, **p < 0.01, ***p < 0.001 between the indicated groups.

    Techniques Used: Transduction, Control, Plasmid Preparation, Over Expression, Staining, Membrane, shRNA, Quantitative RT-PCR, Analysis, Expressing, Western Blot

    Overexpression of TXN2 rescues ARG2-induced mitophagy impairment and cellular senescence in osteoblasts. (A) Venn diagram analysis of mitochondrial oxidative stress-related genes (MOSRGs), mitophagy-related genes (MRGs), osteoporosis-related genes (OSRGs), ARG2-related genes (ARG2RGs), and senescence-related genes (SRGs) retrieved from the GeneCards database. MC3T3-E1 cells were transduced with pLJM1-EGFP (Con) or pLJM1-HA-ARG2 (CMV-ARG2) for ARG2 overexpression. (B) qRT-PCR analysis of the mRNA expression of Cdkn1a, FAS, CAT and TXN2. Senescent MC3T3 E1s were transduced with pLJM1-EGFP as a control (Con) or pLJM1-HA-TXN2 (EF1a-TXN2) to overexpress TXN2. (C) qRT-PCR analysis of mRNA levels of TXN2 overexpression efficiency in OBs. (D) Immunoblotting analysis of TXN2 overexpression efficiency in OBs. The bar graphs on the right show quantifications of protein levels. MC3T3-E1 cells were transduced with pLJM1-EGFP (Con) or pLJM1-HA-ARG2 (CMV-ARG2) for ARG2 overexpression, and subsequently with pLJM1-HA-TXN2 (EF1a-TXN2) to overexpress TXN2. (E) Mitochondrial superoxide was stained with 1 μM MitoSOX. Scale bar = 100 μm. (F) Δψm Assessment by JC-1 staining. Scale bar = 100 μm. (G) Representative TEM images showing mitophagy. Red arrows: mitophagy; White arrows: mitochondria. Scale bar=500 nm. (H) qRT-PCR analysis of the mRNA expression of PINK1 and Parkin. (I) Immunoblotting analysis of p21, p53, ARG2, TXN2, p62 and LC3 protein expression. The bar graphs on the right show quantifications of protein levels. (J) qRT-PCR analysis of the mRNA expression of p21, p53, ARG2 and TXN2. (K) SA-β-gal staining. Bar graphs represent quantifications of SA-β-gal-positive cells. (L) Alizarin Red S staining (ARS) and quantitative analysis of mineral deposition. Scale bar = 100 μm. (M) Alkaline phosphatase (ALP) activity assay. Statistical differences were examined by one-way ANOVA. Data are presented as mean ± SEM. n = 4. ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001 between the indicated groups.
    Figure Legend Snippet: Overexpression of TXN2 rescues ARG2-induced mitophagy impairment and cellular senescence in osteoblasts. (A) Venn diagram analysis of mitochondrial oxidative stress-related genes (MOSRGs), mitophagy-related genes (MRGs), osteoporosis-related genes (OSRGs), ARG2-related genes (ARG2RGs), and senescence-related genes (SRGs) retrieved from the GeneCards database. MC3T3-E1 cells were transduced with pLJM1-EGFP (Con) or pLJM1-HA-ARG2 (CMV-ARG2) for ARG2 overexpression. (B) qRT-PCR analysis of the mRNA expression of Cdkn1a, FAS, CAT and TXN2. Senescent MC3T3 E1s were transduced with pLJM1-EGFP as a control (Con) or pLJM1-HA-TXN2 (EF1a-TXN2) to overexpress TXN2. (C) qRT-PCR analysis of mRNA levels of TXN2 overexpression efficiency in OBs. (D) Immunoblotting analysis of TXN2 overexpression efficiency in OBs. The bar graphs on the right show quantifications of protein levels. MC3T3-E1 cells were transduced with pLJM1-EGFP (Con) or pLJM1-HA-ARG2 (CMV-ARG2) for ARG2 overexpression, and subsequently with pLJM1-HA-TXN2 (EF1a-TXN2) to overexpress TXN2. (E) Mitochondrial superoxide was stained with 1 μM MitoSOX. Scale bar = 100 μm. (F) Δψm Assessment by JC-1 staining. Scale bar = 100 μm. (G) Representative TEM images showing mitophagy. Red arrows: mitophagy; White arrows: mitochondria. Scale bar=500 nm. (H) qRT-PCR analysis of the mRNA expression of PINK1 and Parkin. (I) Immunoblotting analysis of p21, p53, ARG2, TXN2, p62 and LC3 protein expression. The bar graphs on the right show quantifications of protein levels. (J) qRT-PCR analysis of the mRNA expression of p21, p53, ARG2 and TXN2. (K) SA-β-gal staining. Bar graphs represent quantifications of SA-β-gal-positive cells. (L) Alizarin Red S staining (ARS) and quantitative analysis of mineral deposition. Scale bar = 100 μm. (M) Alkaline phosphatase (ALP) activity assay. Statistical differences were examined by one-way ANOVA. Data are presented as mean ± SEM. n = 4. ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001 between the indicated groups.

    Techniques Used: Over Expression, Analysis, Transduction, Quantitative RT-PCR, Expressing, Control, Western Blot, Staining, ALP Activity Assay

    Inhibition of IκBα phosphorylation reverses ARG2-induced downregulation of TXN2 expression, mitophagy and cellular senescence. (A) Venn diagram analysis of TXN2-related genes (TXN2RGs), ARG2-related genes (ARG2RGs), mitophagy-related genes (MRGs), mitochondrial oxidative stress-related genes (MOSRGs), and senescence-related genes (SRGs) retrieved from the GeneCards database. MC3T3-E1 cells were transduced with pLJM1-EGFP (Con) or pLJM1-HA-ARG2 (CMV-ARG2) for ARG2 overexpression. (B) Immunoblotting analysis of IκBα and P-IκBα protein expression. The bar graphs on the right show quantifications of protein levels. MC3T3-E1 cells were transduced with pLJM1-EGFP (Con) or pLJM1-HA-ARG2 (CMV-ARG2) to induce ARG2 overexpression, followed by treatment with Mito-TEMPO (50 μM) or BAY 11-7082 (10 nM) for 24 h. (C) Immunoblotting analysis of IκBα and P-IκBα protein expression. (D) Immunoblotting analysis of p21, p53, ARG2, IκBα, P-IκBα, TXN2, p62 and LC3 protein expression. (E) Mitochondrial superoxide was stained with 1 μM MitoSOX. Scale bar = 100 μm. (F) Δψm assessment by JC-1 staining. Scale bar = 100 μm. (G) Representative transmission electron microscopy (TEM) images showing mitophagy. Red arrows indicate mitophagosomes, and white arrows indicate mitochondria. Scale bar = 500 nm. (H) qRT-PCR analysis of PINK1 and Parkin mRNA expression. (I) qRT-PCR analysis of ARG2, p21, and p53 mRNA expression. (J) Senescence-associated β-galactosidase (SA-β-gal) staining. Quantification of SA-β-gal-positive cells is shown in the accompanying bar graphs. Statistical significance was determined by one-way ANOVA. Data are presented as mean ± SEM (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001 between the indicated groups.
    Figure Legend Snippet: Inhibition of IκBα phosphorylation reverses ARG2-induced downregulation of TXN2 expression, mitophagy and cellular senescence. (A) Venn diagram analysis of TXN2-related genes (TXN2RGs), ARG2-related genes (ARG2RGs), mitophagy-related genes (MRGs), mitochondrial oxidative stress-related genes (MOSRGs), and senescence-related genes (SRGs) retrieved from the GeneCards database. MC3T3-E1 cells were transduced with pLJM1-EGFP (Con) or pLJM1-HA-ARG2 (CMV-ARG2) for ARG2 overexpression. (B) Immunoblotting analysis of IκBα and P-IκBα protein expression. The bar graphs on the right show quantifications of protein levels. MC3T3-E1 cells were transduced with pLJM1-EGFP (Con) or pLJM1-HA-ARG2 (CMV-ARG2) to induce ARG2 overexpression, followed by treatment with Mito-TEMPO (50 μM) or BAY 11-7082 (10 nM) for 24 h. (C) Immunoblotting analysis of IκBα and P-IκBα protein expression. (D) Immunoblotting analysis of p21, p53, ARG2, IκBα, P-IκBα, TXN2, p62 and LC3 protein expression. (E) Mitochondrial superoxide was stained with 1 μM MitoSOX. Scale bar = 100 μm. (F) Δψm assessment by JC-1 staining. Scale bar = 100 μm. (G) Representative transmission electron microscopy (TEM) images showing mitophagy. Red arrows indicate mitophagosomes, and white arrows indicate mitochondria. Scale bar = 500 nm. (H) qRT-PCR analysis of PINK1 and Parkin mRNA expression. (I) qRT-PCR analysis of ARG2, p21, and p53 mRNA expression. (J) Senescence-associated β-galactosidase (SA-β-gal) staining. Quantification of SA-β-gal-positive cells is shown in the accompanying bar graphs. Statistical significance was determined by one-way ANOVA. Data are presented as mean ± SEM (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001 between the indicated groups.

    Techniques Used: Inhibition, Phospho-proteomics, Expressing, Analysis, Transduction, Over Expression, Western Blot, Staining, Transmission Assay, Electron Microscopy, Quantitative RT-PCR

    Disruption of the p65-HDAC9 interaction alleviates ARG2-mediated mitochondrial dysfunction, mitophagy impairment and senescence in osteoblasts. MC3T3-E1 cells were transduced with pLJM1-EGFP (Con) or pLJM1-HA-ARG2 (CMV-ARG2) to induce ARG2 overexpression, followed by treatment with iohexol (25 mg/mL) for 24 h. (A) qRT-PCR analysis of TXN2 mRNA expression. (B) Mitochondrial superoxide production was assessed by MitoSOX staining (1 μM). Scale bar = 100 μm. (C) Mitochondrial membrane potential (Δψm) was evaluated by JC-1 staining. Scale bar = 100 μm. (D) Quantitative JC-1 staining analysis. (E) Representative TEM images showing mitophagy. Red arrows indicate mitophagic vesicles, and white arrows indicate mitochondria. Scale bar = 500 nm. (F) qRT-PCR analysis of PINK1 and Parkin mRNA expression. (G) Immunoblotting analysis of TXN2, p21, p53, ARG2, p62, IκBα, phosphorylated IκBα (P-IκBα), and LC3 protein expression. Quantification of protein levels is shown in the bar graphs on the right. (H) qRT-PCR analysis of p21 and p53 mRNA expression. (I) SA-β-gal staining. Bar graphs represent the quantification of SA-β-gal-positive cells. Scale bar = 100 μm. Statistical significance was determined by one-way ANOVA. Data are presented as mean ± SEM (n = 4). ns, not significant; **p < 0.01, ***p < 0.001 between the indicated groups.
    Figure Legend Snippet: Disruption of the p65-HDAC9 interaction alleviates ARG2-mediated mitochondrial dysfunction, mitophagy impairment and senescence in osteoblasts. MC3T3-E1 cells were transduced with pLJM1-EGFP (Con) or pLJM1-HA-ARG2 (CMV-ARG2) to induce ARG2 overexpression, followed by treatment with iohexol (25 mg/mL) for 24 h. (A) qRT-PCR analysis of TXN2 mRNA expression. (B) Mitochondrial superoxide production was assessed by MitoSOX staining (1 μM). Scale bar = 100 μm. (C) Mitochondrial membrane potential (Δψm) was evaluated by JC-1 staining. Scale bar = 100 μm. (D) Quantitative JC-1 staining analysis. (E) Representative TEM images showing mitophagy. Red arrows indicate mitophagic vesicles, and white arrows indicate mitochondria. Scale bar = 500 nm. (F) qRT-PCR analysis of PINK1 and Parkin mRNA expression. (G) Immunoblotting analysis of TXN2, p21, p53, ARG2, p62, IκBα, phosphorylated IκBα (P-IκBα), and LC3 protein expression. Quantification of protein levels is shown in the bar graphs on the right. (H) qRT-PCR analysis of p21 and p53 mRNA expression. (I) SA-β-gal staining. Bar graphs represent the quantification of SA-β-gal-positive cells. Scale bar = 100 μm. Statistical significance was determined by one-way ANOVA. Data are presented as mean ± SEM (n = 4). ns, not significant; **p < 0.01, ***p < 0.001 between the indicated groups.

    Techniques Used: Disruption, Transduction, Over Expression, Quantitative RT-PCR, Analysis, Expressing, Staining, Membrane, Western Blot

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    Flow Cytometry:

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    Staining:

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    Membrane:

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    Fluorescence:

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    Expressing:

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    Article Snippet: Intracellular superoxide levels were measured using the MitoSOX Red fluorescent probe (HY‐D1055; MCE, USA).. Cells were treated with the MitoSOX Red working solution at 37°C in the dark for 20 min.Cells were treated with the MitoSOX Red working solution at 37°C in the dark for 20 min.

    Article Title: Extracellular vesicles derived from stem cells of human exfoliated deciduous teeth alleviate pulpitis through attenuation of mitochondrial dysfunction
    Article Snippet: The morphology of mitochondria was observed via Mito Tracker red dye (Beyotime) according to the manufacturer’s protocols.The morphology of mitochondria was observed via Mito Tracker red dye (Beyotime) according to the manufacturer’s protocols.. The levels of mtROS were measured by MitoSOX Red fluorescent probe (MedChemExpress, USA).. The cell samples were treated with 4 μM MitoSOX Red for 10 min at 37 °C in the dark, followed by nucleus staining with Hoechst 33,342 (Beyotime).The cell samples were treated with 4 μM MitoSOX Red for 10 min at 37 °C in the dark, followed by nucleus staining with Hoechst 33,342 (Beyotime).

    Article Title: Zinc Overload in Microvessels Contributes to Blood–Brain Barrier Disruption by Activating the JAK2 Pathway After Cerebral Ischemia/Reperfusion
    Article Snippet: Mitochondrial superoxide levels were detected using the MitoSOX Red fluorescent probe (MedChemExpress) [ ].. Briefly, purified mitochondrial (50 μg) isolated from the ischemia hemisphere was stained with MitoSOX Red (at a final concentration of 5 μM) at 37°C for 30 mins.Briefly, purified mitochondrial (50 μg) isolated from the ischemia hemisphere was stained with MitoSOX Red (at a final concentration of 5 μM) at 37°C for 30 mins.

    Article Title: Zinc Overload in Microvessels Contributes to Blood-Brain Barrier Disruption by Activating the JAK2 Pathway After Cerebral Ischemia/Reperfusion.
    Article Snippet: Mitochondrial superoxide levels were detected using the MitoSOX Red fluorescent probe (MedChemExpress) [17].. Briefly, purified mitochondrial (50 μg) isolated from the ischemia hemisphere was stained with MitoSOX Red (at a final concentration of 5 μM) at 37°C for 30 mins.Briefly, purified mitochondrial (50 μg) isolated from the ischemia hemisphere was stained with MitoSOX Red (at a final concentration of 5 μM) at 37°C for 30 mins.

    Activity Assay:

    Article Title: HNRNPC Succinylation Influences the Neurodegeneration of Alzheimer's Disease Through YME1L1 ‐Mediated Mitochondrial Metabolism
    Article Snippet: Intracellular superoxide levels were measured using the MitoSOX Red fluorescent probe (HY‐D1055; MCE, USA).. Cells were treated with the MitoSOX Red working solution at 37°C in the dark for 20 min.Cells were treated with the MitoSOX Red working solution at 37°C in the dark for 20 min.

    Article Title: Extracellular vesicles derived from stem cells of human exfoliated deciduous teeth alleviate pulpitis through attenuation of mitochondrial dysfunction
    Article Snippet: The morphology of mitochondria was observed via Mito Tracker red dye (Beyotime) according to the manufacturer’s protocols.The morphology of mitochondria was observed via Mito Tracker red dye (Beyotime) according to the manufacturer’s protocols.. The levels of mtROS were measured by MitoSOX Red fluorescent probe (MedChemExpress, USA).. The cell samples were treated with 4 μM MitoSOX Red for 10 min at 37 °C in the dark, followed by nucleus staining with Hoechst 33,342 (Beyotime).The cell samples were treated with 4 μM MitoSOX Red for 10 min at 37 °C in the dark, followed by nucleus staining with Hoechst 33,342 (Beyotime).

    Article Title: Zinc Overload in Microvessels Contributes to Blood–Brain Barrier Disruption by Activating the JAK2 Pathway After Cerebral Ischemia/Reperfusion
    Article Snippet: Mitochondrial superoxide levels were detected using the MitoSOX Red fluorescent probe (MedChemExpress) [ ].. Briefly, purified mitochondrial (50 μg) isolated from the ischemia hemisphere was stained with MitoSOX Red (at a final concentration of 5 μM) at 37°C for 30 mins.Briefly, purified mitochondrial (50 μg) isolated from the ischemia hemisphere was stained with MitoSOX Red (at a final concentration of 5 μM) at 37°C for 30 mins.

    Article Title: Zinc Overload in Microvessels Contributes to Blood-Brain Barrier Disruption by Activating the JAK2 Pathway After Cerebral Ischemia/Reperfusion.
    Article Snippet: Mitochondrial superoxide levels were detected using the MitoSOX Red fluorescent probe (MedChemExpress) [17].. Briefly, purified mitochondrial (50 μg) isolated from the ischemia hemisphere was stained with MitoSOX Red (at a final concentration of 5 μM) at 37°C for 30 mins.Briefly, purified mitochondrial (50 μg) isolated from the ischemia hemisphere was stained with MitoSOX Red (at a final concentration of 5 μM) at 37°C for 30 mins.

    Knock-Out:

    Article Title: HNRNPC Succinylation Influences the Neurodegeneration of Alzheimer's Disease Through YME1L1 ‐Mediated Mitochondrial Metabolism
    Article Snippet: Intracellular superoxide levels were measured using the MitoSOX Red fluorescent probe (HY‐D1055; MCE, USA).. Cells were treated with the MitoSOX Red working solution at 37°C in the dark for 20 min.Cells were treated with the MitoSOX Red working solution at 37°C in the dark for 20 min.

    Article Title: Extracellular vesicles derived from stem cells of human exfoliated deciduous teeth alleviate pulpitis through attenuation of mitochondrial dysfunction
    Article Snippet: The morphology of mitochondria was observed via Mito Tracker red dye (Beyotime) according to the manufacturer’s protocols.The morphology of mitochondria was observed via Mito Tracker red dye (Beyotime) according to the manufacturer’s protocols.. The levels of mtROS were measured by MitoSOX Red fluorescent probe (MedChemExpress, USA).. The cell samples were treated with 4 μM MitoSOX Red for 10 min at 37 °C in the dark, followed by nucleus staining with Hoechst 33,342 (Beyotime).The cell samples were treated with 4 μM MitoSOX Red for 10 min at 37 °C in the dark, followed by nucleus staining with Hoechst 33,342 (Beyotime).

    Article Title: Zinc Overload in Microvessels Contributes to Blood–Brain Barrier Disruption by Activating the JAK2 Pathway After Cerebral Ischemia/Reperfusion
    Article Snippet: Mitochondrial superoxide levels were detected using the MitoSOX Red fluorescent probe (MedChemExpress) [ ].. Briefly, purified mitochondrial (50 μg) isolated from the ischemia hemisphere was stained with MitoSOX Red (at a final concentration of 5 μM) at 37°C for 30 mins.Briefly, purified mitochondrial (50 μg) isolated from the ischemia hemisphere was stained with MitoSOX Red (at a final concentration of 5 μM) at 37°C for 30 mins.

    Article Title: Zinc Overload in Microvessels Contributes to Blood-Brain Barrier Disruption by Activating the JAK2 Pathway After Cerebral Ischemia/Reperfusion.
    Article Snippet: Mitochondrial superoxide levels were detected using the MitoSOX Red fluorescent probe (MedChemExpress) [17].. Briefly, purified mitochondrial (50 μg) isolated from the ischemia hemisphere was stained with MitoSOX Red (at a final concentration of 5 μM) at 37°C for 30 mins.Briefly, purified mitochondrial (50 μg) isolated from the ischemia hemisphere was stained with MitoSOX Red (at a final concentration of 5 μM) at 37°C for 30 mins.

    Phospho-proteomics:

    Article Title: HNRNPC Succinylation Influences the Neurodegeneration of Alzheimer's Disease Through YME1L1 ‐Mediated Mitochondrial Metabolism
    Article Snippet: Intracellular superoxide levels were measured using the MitoSOX Red fluorescent probe (HY‐D1055; MCE, USA).. Cells were treated with the MitoSOX Red working solution at 37°C in the dark for 20 min.Cells were treated with the MitoSOX Red working solution at 37°C in the dark for 20 min.

    Article Title: Extracellular vesicles derived from stem cells of human exfoliated deciduous teeth alleviate pulpitis through attenuation of mitochondrial dysfunction
    Article Snippet: The morphology of mitochondria was observed via Mito Tracker red dye (Beyotime) according to the manufacturer’s protocols.The morphology of mitochondria was observed via Mito Tracker red dye (Beyotime) according to the manufacturer’s protocols.. The levels of mtROS were measured by MitoSOX Red fluorescent probe (MedChemExpress, USA).. The cell samples were treated with 4 μM MitoSOX Red for 10 min at 37 °C in the dark, followed by nucleus staining with Hoechst 33,342 (Beyotime).The cell samples were treated with 4 μM MitoSOX Red for 10 min at 37 °C in the dark, followed by nucleus staining with Hoechst 33,342 (Beyotime).

    Article Title: Zinc Overload in Microvessels Contributes to Blood–Brain Barrier Disruption by Activating the JAK2 Pathway After Cerebral Ischemia/Reperfusion
    Article Snippet: Mitochondrial superoxide levels were detected using the MitoSOX Red fluorescent probe (MedChemExpress) [ ].. Briefly, purified mitochondrial (50 μg) isolated from the ischemia hemisphere was stained with MitoSOX Red (at a final concentration of 5 μM) at 37°C for 30 mins.Briefly, purified mitochondrial (50 μg) isolated from the ischemia hemisphere was stained with MitoSOX Red (at a final concentration of 5 μM) at 37°C for 30 mins.

    Article Title: Zinc Overload in Microvessels Contributes to Blood-Brain Barrier Disruption by Activating the JAK2 Pathway After Cerebral Ischemia/Reperfusion.
    Article Snippet: Mitochondrial superoxide levels were detected using the MitoSOX Red fluorescent probe (MedChemExpress) [17].. Briefly, purified mitochondrial (50 μg) isolated from the ischemia hemisphere was stained with MitoSOX Red (at a final concentration of 5 μM) at 37°C for 30 mins.Briefly, purified mitochondrial (50 μg) isolated from the ischemia hemisphere was stained with MitoSOX Red (at a final concentration of 5 μM) at 37°C for 30 mins.

    Western Blot:

    Article Title: HNRNPC Succinylation Influences the Neurodegeneration of Alzheimer's Disease Through YME1L1 ‐Mediated Mitochondrial Metabolism
    Article Snippet: Intracellular superoxide levels were measured using the MitoSOX Red fluorescent probe (HY‐D1055; MCE, USA).. Cells were treated with the MitoSOX Red working solution at 37°C in the dark for 20 min.Cells were treated with the MitoSOX Red working solution at 37°C in the dark for 20 min.

    Article Title: Extracellular vesicles derived from stem cells of human exfoliated deciduous teeth alleviate pulpitis through attenuation of mitochondrial dysfunction
    Article Snippet: The morphology of mitochondria was observed via Mito Tracker red dye (Beyotime) according to the manufacturer’s protocols.The morphology of mitochondria was observed via Mito Tracker red dye (Beyotime) according to the manufacturer’s protocols.. The levels of mtROS were measured by MitoSOX Red fluorescent probe (MedChemExpress, USA).. The cell samples were treated with 4 μM MitoSOX Red for 10 min at 37 °C in the dark, followed by nucleus staining with Hoechst 33,342 (Beyotime).The cell samples were treated with 4 μM MitoSOX Red for 10 min at 37 °C in the dark, followed by nucleus staining with Hoechst 33,342 (Beyotime).

    Article Title: Zinc Overload in Microvessels Contributes to Blood–Brain Barrier Disruption by Activating the JAK2 Pathway After Cerebral Ischemia/Reperfusion
    Article Snippet: Mitochondrial superoxide levels were detected using the MitoSOX Red fluorescent probe (MedChemExpress) [ ].. Briefly, purified mitochondrial (50 μg) isolated from the ischemia hemisphere was stained with MitoSOX Red (at a final concentration of 5 μM) at 37°C for 30 mins.Briefly, purified mitochondrial (50 μg) isolated from the ischemia hemisphere was stained with MitoSOX Red (at a final concentration of 5 μM) at 37°C for 30 mins.

    Article Title: Zinc Overload in Microvessels Contributes to Blood-Brain Barrier Disruption by Activating the JAK2 Pathway After Cerebral Ischemia/Reperfusion.
    Article Snippet: Mitochondrial superoxide levels were detected using the MitoSOX Red fluorescent probe (MedChemExpress) [17].. Briefly, purified mitochondrial (50 μg) isolated from the ischemia hemisphere was stained with MitoSOX Red (at a final concentration of 5 μM) at 37°C for 30 mins.Briefly, purified mitochondrial (50 μg) isolated from the ischemia hemisphere was stained with MitoSOX Red (at a final concentration of 5 μM) at 37°C for 30 mins.

    Isolation:

    Article Title: HNRNPC Succinylation Influences the Neurodegeneration of Alzheimer's Disease Through YME1L1 ‐Mediated Mitochondrial Metabolism
    Article Snippet: Intracellular superoxide levels were measured using the MitoSOX Red fluorescent probe (HY‐D1055; MCE, USA).. Cells were treated with the MitoSOX Red working solution at 37°C in the dark for 20 min.Cells were treated with the MitoSOX Red working solution at 37°C in the dark for 20 min.

    Article Title: Extracellular vesicles derived from stem cells of human exfoliated deciduous teeth alleviate pulpitis through attenuation of mitochondrial dysfunction
    Article Snippet: The morphology of mitochondria was observed via Mito Tracker red dye (Beyotime) according to the manufacturer’s protocols.The morphology of mitochondria was observed via Mito Tracker red dye (Beyotime) according to the manufacturer’s protocols.. The levels of mtROS were measured by MitoSOX Red fluorescent probe (MedChemExpress, USA).. The cell samples were treated with 4 μM MitoSOX Red for 10 min at 37 °C in the dark, followed by nucleus staining with Hoechst 33,342 (Beyotime).The cell samples were treated with 4 μM MitoSOX Red for 10 min at 37 °C in the dark, followed by nucleus staining with Hoechst 33,342 (Beyotime).

    Article Title: Zinc Overload in Microvessels Contributes to Blood–Brain Barrier Disruption by Activating the JAK2 Pathway After Cerebral Ischemia/Reperfusion
    Article Snippet: Mitochondrial superoxide levels were detected using the MitoSOX Red fluorescent probe (MedChemExpress) [ ].. Briefly, purified mitochondrial (50 μg) isolated from the ischemia hemisphere was stained with MitoSOX Red (at a final concentration of 5 μM) at 37°C for 30 mins.Briefly, purified mitochondrial (50 μg) isolated from the ischemia hemisphere was stained with MitoSOX Red (at a final concentration of 5 μM) at 37°C for 30 mins.

    Article Title: Zinc Overload in Microvessels Contributes to Blood-Brain Barrier Disruption by Activating the JAK2 Pathway After Cerebral Ischemia/Reperfusion.
    Article Snippet: Mitochondrial superoxide levels were detected using the MitoSOX Red fluorescent probe (MedChemExpress) [17].. Briefly, purified mitochondrial (50 μg) isolated from the ischemia hemisphere was stained with MitoSOX Red (at a final concentration of 5 μM) at 37°C for 30 mins.Briefly, purified mitochondrial (50 μg) isolated from the ischemia hemisphere was stained with MitoSOX Red (at a final concentration of 5 μM) at 37°C for 30 mins.

    Control:

    Article Title: HNRNPC Succinylation Influences the Neurodegeneration of Alzheimer's Disease Through YME1L1 ‐Mediated Mitochondrial Metabolism
    Article Snippet: Intracellular superoxide levels were measured using the MitoSOX Red fluorescent probe (HY‐D1055; MCE, USA).. Cells were treated with the MitoSOX Red working solution at 37°C in the dark for 20 min.Cells were treated with the MitoSOX Red working solution at 37°C in the dark for 20 min.

    Article Title: Extracellular vesicles derived from stem cells of human exfoliated deciduous teeth alleviate pulpitis through attenuation of mitochondrial dysfunction
    Article Snippet: The morphology of mitochondria was observed via Mito Tracker red dye (Beyotime) according to the manufacturer’s protocols.The morphology of mitochondria was observed via Mito Tracker red dye (Beyotime) according to the manufacturer’s protocols.. The levels of mtROS were measured by MitoSOX Red fluorescent probe (MedChemExpress, USA).. The cell samples were treated with 4 μM MitoSOX Red for 10 min at 37 °C in the dark, followed by nucleus staining with Hoechst 33,342 (Beyotime).The cell samples were treated with 4 μM MitoSOX Red for 10 min at 37 °C in the dark, followed by nucleus staining with Hoechst 33,342 (Beyotime).

    Article Title: Zinc Overload in Microvessels Contributes to Blood–Brain Barrier Disruption by Activating the JAK2 Pathway After Cerebral Ischemia/Reperfusion
    Article Snippet: Mitochondrial superoxide levels were detected using the MitoSOX Red fluorescent probe (MedChemExpress) [ ].. Briefly, purified mitochondrial (50 μg) isolated from the ischemia hemisphere was stained with MitoSOX Red (at a final concentration of 5 μM) at 37°C for 30 mins.Briefly, purified mitochondrial (50 μg) isolated from the ischemia hemisphere was stained with MitoSOX Red (at a final concentration of 5 μM) at 37°C for 30 mins.

    Article Title: Zinc Overload in Microvessels Contributes to Blood-Brain Barrier Disruption by Activating the JAK2 Pathway After Cerebral Ischemia/Reperfusion.
    Article Snippet: Mitochondrial superoxide levels were detected using the MitoSOX Red fluorescent probe (MedChemExpress) [17].. Briefly, purified mitochondrial (50 μg) isolated from the ischemia hemisphere was stained with MitoSOX Red (at a final concentration of 5 μM) at 37°C for 30 mins.Briefly, purified mitochondrial (50 μg) isolated from the ischemia hemisphere was stained with MitoSOX Red (at a final concentration of 5 μM) at 37°C for 30 mins.

    Saline:

    Article Title: HNRNPC Succinylation Influences the Neurodegeneration of Alzheimer's Disease Through YME1L1 ‐Mediated Mitochondrial Metabolism
    Article Snippet: Intracellular superoxide levels were measured using the MitoSOX Red fluorescent probe (HY‐D1055; MCE, USA).. Cells were treated with the MitoSOX Red working solution at 37°C in the dark for 20 min.Cells were treated with the MitoSOX Red working solution at 37°C in the dark for 20 min.

    Article Title: Extracellular vesicles derived from stem cells of human exfoliated deciduous teeth alleviate pulpitis through attenuation of mitochondrial dysfunction
    Article Snippet: The morphology of mitochondria was observed via Mito Tracker red dye (Beyotime) according to the manufacturer’s protocols.The morphology of mitochondria was observed via Mito Tracker red dye (Beyotime) according to the manufacturer’s protocols.. The levels of mtROS were measured by MitoSOX Red fluorescent probe (MedChemExpress, USA).. The cell samples were treated with 4 μM MitoSOX Red for 10 min at 37 °C in the dark, followed by nucleus staining with Hoechst 33,342 (Beyotime).The cell samples were treated with 4 μM MitoSOX Red for 10 min at 37 °C in the dark, followed by nucleus staining with Hoechst 33,342 (Beyotime).

    Article Title: Zinc Overload in Microvessels Contributes to Blood–Brain Barrier Disruption by Activating the JAK2 Pathway After Cerebral Ischemia/Reperfusion
    Article Snippet: Mitochondrial superoxide levels were detected using the MitoSOX Red fluorescent probe (MedChemExpress) [ ].. Briefly, purified mitochondrial (50 μg) isolated from the ischemia hemisphere was stained with MitoSOX Red (at a final concentration of 5 μM) at 37°C for 30 mins.Briefly, purified mitochondrial (50 μg) isolated from the ischemia hemisphere was stained with MitoSOX Red (at a final concentration of 5 μM) at 37°C for 30 mins.

    Article Title: Zinc Overload in Microvessels Contributes to Blood-Brain Barrier Disruption by Activating the JAK2 Pathway After Cerebral Ischemia/Reperfusion.
    Article Snippet: Mitochondrial superoxide levels were detected using the MitoSOX Red fluorescent probe (MedChemExpress) [17].. Briefly, purified mitochondrial (50 μg) isolated from the ischemia hemisphere was stained with MitoSOX Red (at a final concentration of 5 μM) at 37°C for 30 mins.Briefly, purified mitochondrial (50 μg) isolated from the ischemia hemisphere was stained with MitoSOX Red (at a final concentration of 5 μM) at 37°C for 30 mins.



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    NAC alleviates ARG2-induced mitophagy suppression and cellular senescence in osteoblasts. MC3T3-E1 cells were transduced with pLJM1-EGFP control vector (Con) or pLJM1-HA-ARG2 (CMV-ARG2) to induce ARG2 overexpression. (A) Mitochondrial superoxide levels were assessed by MitoSOX staining (1 μM). (B) Mitochondrial membrane potential (Δψm) was evaluated by JC-1 staining. MC3T3-E1 cells were transduced with pLKO.1-control (Con) or pLKO.1-ARG2 shRNA (sh-ARG2) lentivirus for 48 h, followed by treatment with H₂O₂ (200 μM) for 24 h. (C) Mitochondrial superoxide levels were determined by MitoSOX staining (1 μM). (D) Δψm was assessed by JC-1 staining. For rescue experiments, MC3T3-E1 cells transduced with pLJM1-EGFP (Con) or pLJM1-HA-ARG2 (CMV-ARG2) were treated with N-acetylcysteine (NAC, 500 μM) for 24 h. (E) Mitochondrial superoxide levels were evaluated by MitoSOX staining (1 μM). (F) Δψm was assessed by JC-1 staining. (G) qRT-PCR analysis of PINK1 and Parkin mRNA expression. (H) Immunoblot analysis of p53, p21, p62, and LC3 protein expression. Quantitative analyses of protein levels are shown in the bar graphs on the right. (I) Senescence-associated β-galactosidase (SA-β-gal) staining. Bar graphs represent the quantification of SA-β-gal-positive cells. Statistical significance was determined by one-way ANOVA. Data are presented as mean ± SEM (n = 4). Scale bar = 100 μm. *p < 0.05, **p < 0.01, ***p < 0.001 between the indicated groups.

    Journal: Theranostics

    Article Title: ARG2 drives osteoblast senescence by repressing mitophagy via the IκB–NF-κB–TXN2 signaling axis: implications for age-associated osteoporosis

    doi: 10.7150/thno.129818

    Figure Lengend Snippet: NAC alleviates ARG2-induced mitophagy suppression and cellular senescence in osteoblasts. MC3T3-E1 cells were transduced with pLJM1-EGFP control vector (Con) or pLJM1-HA-ARG2 (CMV-ARG2) to induce ARG2 overexpression. (A) Mitochondrial superoxide levels were assessed by MitoSOX staining (1 μM). (B) Mitochondrial membrane potential (Δψm) was evaluated by JC-1 staining. MC3T3-E1 cells were transduced with pLKO.1-control (Con) or pLKO.1-ARG2 shRNA (sh-ARG2) lentivirus for 48 h, followed by treatment with H₂O₂ (200 μM) for 24 h. (C) Mitochondrial superoxide levels were determined by MitoSOX staining (1 μM). (D) Δψm was assessed by JC-1 staining. For rescue experiments, MC3T3-E1 cells transduced with pLJM1-EGFP (Con) or pLJM1-HA-ARG2 (CMV-ARG2) were treated with N-acetylcysteine (NAC, 500 μM) for 24 h. (E) Mitochondrial superoxide levels were evaluated by MitoSOX staining (1 μM). (F) Δψm was assessed by JC-1 staining. (G) qRT-PCR analysis of PINK1 and Parkin mRNA expression. (H) Immunoblot analysis of p53, p21, p62, and LC3 protein expression. Quantitative analyses of protein levels are shown in the bar graphs on the right. (I) Senescence-associated β-galactosidase (SA-β-gal) staining. Bar graphs represent the quantification of SA-β-gal-positive cells. Statistical significance was determined by one-way ANOVA. Data are presented as mean ± SEM (n = 4). Scale bar = 100 μm. *p < 0.05, **p < 0.01, ***p < 0.001 between the indicated groups.

    Article Snippet: Mitochondrial reactive oxygen species (ROS) production was detected using the MitoSOX fluorescent probe (MedChemExpress, Shanghai, China).

    Techniques: Transduction, Control, Plasmid Preparation, Over Expression, Staining, Membrane, shRNA, Quantitative RT-PCR, Analysis, Expressing, Western Blot

    Overexpression of TXN2 rescues ARG2-induced mitophagy impairment and cellular senescence in osteoblasts. (A) Venn diagram analysis of mitochondrial oxidative stress-related genes (MOSRGs), mitophagy-related genes (MRGs), osteoporosis-related genes (OSRGs), ARG2-related genes (ARG2RGs), and senescence-related genes (SRGs) retrieved from the GeneCards database. MC3T3-E1 cells were transduced with pLJM1-EGFP (Con) or pLJM1-HA-ARG2 (CMV-ARG2) for ARG2 overexpression. (B) qRT-PCR analysis of the mRNA expression of Cdkn1a, FAS, CAT and TXN2. Senescent MC3T3 E1s were transduced with pLJM1-EGFP as a control (Con) or pLJM1-HA-TXN2 (EF1a-TXN2) to overexpress TXN2. (C) qRT-PCR analysis of mRNA levels of TXN2 overexpression efficiency in OBs. (D) Immunoblotting analysis of TXN2 overexpression efficiency in OBs. The bar graphs on the right show quantifications of protein levels. MC3T3-E1 cells were transduced with pLJM1-EGFP (Con) or pLJM1-HA-ARG2 (CMV-ARG2) for ARG2 overexpression, and subsequently with pLJM1-HA-TXN2 (EF1a-TXN2) to overexpress TXN2. (E) Mitochondrial superoxide was stained with 1 μM MitoSOX. Scale bar = 100 μm. (F) Δψm Assessment by JC-1 staining. Scale bar = 100 μm. (G) Representative TEM images showing mitophagy. Red arrows: mitophagy; White arrows: mitochondria. Scale bar=500 nm. (H) qRT-PCR analysis of the mRNA expression of PINK1 and Parkin. (I) Immunoblotting analysis of p21, p53, ARG2, TXN2, p62 and LC3 protein expression. The bar graphs on the right show quantifications of protein levels. (J) qRT-PCR analysis of the mRNA expression of p21, p53, ARG2 and TXN2. (K) SA-β-gal staining. Bar graphs represent quantifications of SA-β-gal-positive cells. (L) Alizarin Red S staining (ARS) and quantitative analysis of mineral deposition. Scale bar = 100 μm. (M) Alkaline phosphatase (ALP) activity assay. Statistical differences were examined by one-way ANOVA. Data are presented as mean ± SEM. n = 4. ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001 between the indicated groups.

    Journal: Theranostics

    Article Title: ARG2 drives osteoblast senescence by repressing mitophagy via the IκB–NF-κB–TXN2 signaling axis: implications for age-associated osteoporosis

    doi: 10.7150/thno.129818

    Figure Lengend Snippet: Overexpression of TXN2 rescues ARG2-induced mitophagy impairment and cellular senescence in osteoblasts. (A) Venn diagram analysis of mitochondrial oxidative stress-related genes (MOSRGs), mitophagy-related genes (MRGs), osteoporosis-related genes (OSRGs), ARG2-related genes (ARG2RGs), and senescence-related genes (SRGs) retrieved from the GeneCards database. MC3T3-E1 cells were transduced with pLJM1-EGFP (Con) or pLJM1-HA-ARG2 (CMV-ARG2) for ARG2 overexpression. (B) qRT-PCR analysis of the mRNA expression of Cdkn1a, FAS, CAT and TXN2. Senescent MC3T3 E1s were transduced with pLJM1-EGFP as a control (Con) or pLJM1-HA-TXN2 (EF1a-TXN2) to overexpress TXN2. (C) qRT-PCR analysis of mRNA levels of TXN2 overexpression efficiency in OBs. (D) Immunoblotting analysis of TXN2 overexpression efficiency in OBs. The bar graphs on the right show quantifications of protein levels. MC3T3-E1 cells were transduced with pLJM1-EGFP (Con) or pLJM1-HA-ARG2 (CMV-ARG2) for ARG2 overexpression, and subsequently with pLJM1-HA-TXN2 (EF1a-TXN2) to overexpress TXN2. (E) Mitochondrial superoxide was stained with 1 μM MitoSOX. Scale bar = 100 μm. (F) Δψm Assessment by JC-1 staining. Scale bar = 100 μm. (G) Representative TEM images showing mitophagy. Red arrows: mitophagy; White arrows: mitochondria. Scale bar=500 nm. (H) qRT-PCR analysis of the mRNA expression of PINK1 and Parkin. (I) Immunoblotting analysis of p21, p53, ARG2, TXN2, p62 and LC3 protein expression. The bar graphs on the right show quantifications of protein levels. (J) qRT-PCR analysis of the mRNA expression of p21, p53, ARG2 and TXN2. (K) SA-β-gal staining. Bar graphs represent quantifications of SA-β-gal-positive cells. (L) Alizarin Red S staining (ARS) and quantitative analysis of mineral deposition. Scale bar = 100 μm. (M) Alkaline phosphatase (ALP) activity assay. Statistical differences were examined by one-way ANOVA. Data are presented as mean ± SEM. n = 4. ns: not significant, *p < 0.05, **p < 0.01, ***p < 0.001 between the indicated groups.

    Article Snippet: Mitochondrial reactive oxygen species (ROS) production was detected using the MitoSOX fluorescent probe (MedChemExpress, Shanghai, China).

    Techniques: Over Expression, Analysis, Transduction, Quantitative RT-PCR, Expressing, Control, Western Blot, Staining, ALP Activity Assay

    Inhibition of IκBα phosphorylation reverses ARG2-induced downregulation of TXN2 expression, mitophagy and cellular senescence. (A) Venn diagram analysis of TXN2-related genes (TXN2RGs), ARG2-related genes (ARG2RGs), mitophagy-related genes (MRGs), mitochondrial oxidative stress-related genes (MOSRGs), and senescence-related genes (SRGs) retrieved from the GeneCards database. MC3T3-E1 cells were transduced with pLJM1-EGFP (Con) or pLJM1-HA-ARG2 (CMV-ARG2) for ARG2 overexpression. (B) Immunoblotting analysis of IκBα and P-IκBα protein expression. The bar graphs on the right show quantifications of protein levels. MC3T3-E1 cells were transduced with pLJM1-EGFP (Con) or pLJM1-HA-ARG2 (CMV-ARG2) to induce ARG2 overexpression, followed by treatment with Mito-TEMPO (50 μM) or BAY 11-7082 (10 nM) for 24 h. (C) Immunoblotting analysis of IκBα and P-IκBα protein expression. (D) Immunoblotting analysis of p21, p53, ARG2, IκBα, P-IκBα, TXN2, p62 and LC3 protein expression. (E) Mitochondrial superoxide was stained with 1 μM MitoSOX. Scale bar = 100 μm. (F) Δψm assessment by JC-1 staining. Scale bar = 100 μm. (G) Representative transmission electron microscopy (TEM) images showing mitophagy. Red arrows indicate mitophagosomes, and white arrows indicate mitochondria. Scale bar = 500 nm. (H) qRT-PCR analysis of PINK1 and Parkin mRNA expression. (I) qRT-PCR analysis of ARG2, p21, and p53 mRNA expression. (J) Senescence-associated β-galactosidase (SA-β-gal) staining. Quantification of SA-β-gal-positive cells is shown in the accompanying bar graphs. Statistical significance was determined by one-way ANOVA. Data are presented as mean ± SEM (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001 between the indicated groups.

    Journal: Theranostics

    Article Title: ARG2 drives osteoblast senescence by repressing mitophagy via the IκB–NF-κB–TXN2 signaling axis: implications for age-associated osteoporosis

    doi: 10.7150/thno.129818

    Figure Lengend Snippet: Inhibition of IκBα phosphorylation reverses ARG2-induced downregulation of TXN2 expression, mitophagy and cellular senescence. (A) Venn diagram analysis of TXN2-related genes (TXN2RGs), ARG2-related genes (ARG2RGs), mitophagy-related genes (MRGs), mitochondrial oxidative stress-related genes (MOSRGs), and senescence-related genes (SRGs) retrieved from the GeneCards database. MC3T3-E1 cells were transduced with pLJM1-EGFP (Con) or pLJM1-HA-ARG2 (CMV-ARG2) for ARG2 overexpression. (B) Immunoblotting analysis of IκBα and P-IκBα protein expression. The bar graphs on the right show quantifications of protein levels. MC3T3-E1 cells were transduced with pLJM1-EGFP (Con) or pLJM1-HA-ARG2 (CMV-ARG2) to induce ARG2 overexpression, followed by treatment with Mito-TEMPO (50 μM) or BAY 11-7082 (10 nM) for 24 h. (C) Immunoblotting analysis of IκBα and P-IκBα protein expression. (D) Immunoblotting analysis of p21, p53, ARG2, IκBα, P-IκBα, TXN2, p62 and LC3 protein expression. (E) Mitochondrial superoxide was stained with 1 μM MitoSOX. Scale bar = 100 μm. (F) Δψm assessment by JC-1 staining. Scale bar = 100 μm. (G) Representative transmission electron microscopy (TEM) images showing mitophagy. Red arrows indicate mitophagosomes, and white arrows indicate mitochondria. Scale bar = 500 nm. (H) qRT-PCR analysis of PINK1 and Parkin mRNA expression. (I) qRT-PCR analysis of ARG2, p21, and p53 mRNA expression. (J) Senescence-associated β-galactosidase (SA-β-gal) staining. Quantification of SA-β-gal-positive cells is shown in the accompanying bar graphs. Statistical significance was determined by one-way ANOVA. Data are presented as mean ± SEM (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001 between the indicated groups.

    Article Snippet: Mitochondrial reactive oxygen species (ROS) production was detected using the MitoSOX fluorescent probe (MedChemExpress, Shanghai, China).

    Techniques: Inhibition, Phospho-proteomics, Expressing, Analysis, Transduction, Over Expression, Western Blot, Staining, Transmission Assay, Electron Microscopy, Quantitative RT-PCR

    Disruption of the p65-HDAC9 interaction alleviates ARG2-mediated mitochondrial dysfunction, mitophagy impairment and senescence in osteoblasts. MC3T3-E1 cells were transduced with pLJM1-EGFP (Con) or pLJM1-HA-ARG2 (CMV-ARG2) to induce ARG2 overexpression, followed by treatment with iohexol (25 mg/mL) for 24 h. (A) qRT-PCR analysis of TXN2 mRNA expression. (B) Mitochondrial superoxide production was assessed by MitoSOX staining (1 μM). Scale bar = 100 μm. (C) Mitochondrial membrane potential (Δψm) was evaluated by JC-1 staining. Scale bar = 100 μm. (D) Quantitative JC-1 staining analysis. (E) Representative TEM images showing mitophagy. Red arrows indicate mitophagic vesicles, and white arrows indicate mitochondria. Scale bar = 500 nm. (F) qRT-PCR analysis of PINK1 and Parkin mRNA expression. (G) Immunoblotting analysis of TXN2, p21, p53, ARG2, p62, IκBα, phosphorylated IκBα (P-IκBα), and LC3 protein expression. Quantification of protein levels is shown in the bar graphs on the right. (H) qRT-PCR analysis of p21 and p53 mRNA expression. (I) SA-β-gal staining. Bar graphs represent the quantification of SA-β-gal-positive cells. Scale bar = 100 μm. Statistical significance was determined by one-way ANOVA. Data are presented as mean ± SEM (n = 4). ns, not significant; **p < 0.01, ***p < 0.001 between the indicated groups.

    Journal: Theranostics

    Article Title: ARG2 drives osteoblast senescence by repressing mitophagy via the IκB–NF-κB–TXN2 signaling axis: implications for age-associated osteoporosis

    doi: 10.7150/thno.129818

    Figure Lengend Snippet: Disruption of the p65-HDAC9 interaction alleviates ARG2-mediated mitochondrial dysfunction, mitophagy impairment and senescence in osteoblasts. MC3T3-E1 cells were transduced with pLJM1-EGFP (Con) or pLJM1-HA-ARG2 (CMV-ARG2) to induce ARG2 overexpression, followed by treatment with iohexol (25 mg/mL) for 24 h. (A) qRT-PCR analysis of TXN2 mRNA expression. (B) Mitochondrial superoxide production was assessed by MitoSOX staining (1 μM). Scale bar = 100 μm. (C) Mitochondrial membrane potential (Δψm) was evaluated by JC-1 staining. Scale bar = 100 μm. (D) Quantitative JC-1 staining analysis. (E) Representative TEM images showing mitophagy. Red arrows indicate mitophagic vesicles, and white arrows indicate mitochondria. Scale bar = 500 nm. (F) qRT-PCR analysis of PINK1 and Parkin mRNA expression. (G) Immunoblotting analysis of TXN2, p21, p53, ARG2, p62, IκBα, phosphorylated IκBα (P-IκBα), and LC3 protein expression. Quantification of protein levels is shown in the bar graphs on the right. (H) qRT-PCR analysis of p21 and p53 mRNA expression. (I) SA-β-gal staining. Bar graphs represent the quantification of SA-β-gal-positive cells. Scale bar = 100 μm. Statistical significance was determined by one-way ANOVA. Data are presented as mean ± SEM (n = 4). ns, not significant; **p < 0.01, ***p < 0.001 between the indicated groups.

    Article Snippet: Mitochondrial reactive oxygen species (ROS) production was detected using the MitoSOX fluorescent probe (MedChemExpress, Shanghai, China).

    Techniques: Disruption, Transduction, Over Expression, Quantitative RT-PCR, Analysis, Expressing, Staining, Membrane, Western Blot

    YME1L1 promotes neuronal mitochondrial damage after AD. (A) CCK8 assay for cell viability. (B) Flow cytometry for apoptosis detection. (C) TEM for mitochondrial assessment (The green arrow indicates normal mitochondria, and the red arrow indicates damaged mitochondria). (D) ATP level measurement. (E) JC‐1 staining for cellular mitochondrial membrane potential measurement. (F) MitoSOX fluorescence staining for cellular mitochondrial ROS analysis. n = 3. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

    Journal: Aging Cell

    Article Title: HNRNPC Succinylation Influences the Neurodegeneration of Alzheimer's Disease Through YME1L1 ‐Mediated Mitochondrial Metabolism

    doi: 10.1111/acel.70646

    Figure Lengend Snippet: YME1L1 promotes neuronal mitochondrial damage after AD. (A) CCK8 assay for cell viability. (B) Flow cytometry for apoptosis detection. (C) TEM for mitochondrial assessment (The green arrow indicates normal mitochondria, and the red arrow indicates damaged mitochondria). (D) ATP level measurement. (E) JC‐1 staining for cellular mitochondrial membrane potential measurement. (F) MitoSOX fluorescence staining for cellular mitochondrial ROS analysis. n = 3. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

    Article Snippet: Intracellular superoxide levels were measured using the MitoSOX Red fluorescent probe (HY‐D1055; MCE, USA).

    Techniques: CCK-8 Assay, Flow Cytometry, Staining, Membrane, Fluorescence

    YME1L1 induces neuronal mitochondrial damage and neuronal death following AD through OPA1 hydrolysis. (A) WB detection of OPA1 expression in hippocampal tissue ( n = 6). (B) WB detection of OPA1 expression. (C) CCK8 assay for cell viability. (D) Flow cytometry for apoptosis detection. (E) TEM for mitochondrial detection (The red arrow indicates damaged mitochondria). (F) ATP level detection. (G) JC‐1 staining for measuring mitochondrial membrane potential. (H) MitoSOX fluorescence staining for analyzing mitochondrial ROS. n = 3. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

    Journal: Aging Cell

    Article Title: HNRNPC Succinylation Influences the Neurodegeneration of Alzheimer's Disease Through YME1L1 ‐Mediated Mitochondrial Metabolism

    doi: 10.1111/acel.70646

    Figure Lengend Snippet: YME1L1 induces neuronal mitochondrial damage and neuronal death following AD through OPA1 hydrolysis. (A) WB detection of OPA1 expression in hippocampal tissue ( n = 6). (B) WB detection of OPA1 expression. (C) CCK8 assay for cell viability. (D) Flow cytometry for apoptosis detection. (E) TEM for mitochondrial detection (The red arrow indicates damaged mitochondria). (F) ATP level detection. (G) JC‐1 staining for measuring mitochondrial membrane potential. (H) MitoSOX fluorescence staining for analyzing mitochondrial ROS. n = 3. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

    Article Snippet: Intracellular superoxide levels were measured using the MitoSOX Red fluorescent probe (HY‐D1055; MCE, USA).

    Techniques: Expressing, CCK-8 Assay, Flow Cytometry, Staining, Membrane, Fluorescence

    HNRNPC succinylation modifies neuronal mitochondrial damage and neuronal death following AD via YME1L1. (A) PCR and WB detection of YME1L1, WB detection of OPA1. (B) CCK8 analysis of cell activity. (C) Flow cytometry to detect cell apoptosis. (D) TEM detection of mitochondria (The green arrow points to normal mitochondria, and the red arrow points to damaged mitochondria). (E) ATP level detection. (F) JC‐1 staining was used to measure the mitochondrial membrane potential of cells. (G) MitoSOX fluorescent staining analysis of mitochondrial ROS in cells. n = 3. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

    Journal: Aging Cell

    Article Title: HNRNPC Succinylation Influences the Neurodegeneration of Alzheimer's Disease Through YME1L1 ‐Mediated Mitochondrial Metabolism

    doi: 10.1111/acel.70646

    Figure Lengend Snippet: HNRNPC succinylation modifies neuronal mitochondrial damage and neuronal death following AD via YME1L1. (A) PCR and WB detection of YME1L1, WB detection of OPA1. (B) CCK8 analysis of cell activity. (C) Flow cytometry to detect cell apoptosis. (D) TEM detection of mitochondria (The green arrow points to normal mitochondria, and the red arrow points to damaged mitochondria). (E) ATP level detection. (F) JC‐1 staining was used to measure the mitochondrial membrane potential of cells. (G) MitoSOX fluorescent staining analysis of mitochondrial ROS in cells. n = 3. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

    Article Snippet: Intracellular superoxide levels were measured using the MitoSOX Red fluorescent probe (HY‐D1055; MCE, USA).

    Techniques: Activity Assay, Flow Cytometry, Staining, Membrane

    SIRT5 affects neuronal mitochondrial function by desuccinylating HNRNPC. (A) WB detection of YME1L1 and OPA1 expression. (B) CCK8 analysis of cell activity. (C) Flow cytometry detection of cell apoptosis. (D) TEM detection of mitochondrial morphology (The green arrow points to normal mitochondria, and the red arrow points to damaged mitochondria). (E) ATP level detection. (F) JC‐1 staining to measure cell mitochondrial membrane potential. (G) MitoSOX fluorescent staining analysis of mitochondrial ROS in cells. n = 3. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns stands for no significant difference.

    Journal: Aging Cell

    Article Title: HNRNPC Succinylation Influences the Neurodegeneration of Alzheimer's Disease Through YME1L1 ‐Mediated Mitochondrial Metabolism

    doi: 10.1111/acel.70646

    Figure Lengend Snippet: SIRT5 affects neuronal mitochondrial function by desuccinylating HNRNPC. (A) WB detection of YME1L1 and OPA1 expression. (B) CCK8 analysis of cell activity. (C) Flow cytometry detection of cell apoptosis. (D) TEM detection of mitochondrial morphology (The green arrow points to normal mitochondria, and the red arrow points to damaged mitochondria). (E) ATP level detection. (F) JC‐1 staining to measure cell mitochondrial membrane potential. (G) MitoSOX fluorescent staining analysis of mitochondrial ROS in cells. n = 3. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001; ns stands for no significant difference.

    Article Snippet: Intracellular superoxide levels were measured using the MitoSOX Red fluorescent probe (HY‐D1055; MCE, USA).

    Techniques: Expressing, Activity Assay, Flow Cytometry, Staining, Membrane