recombinant pad4 adenovirus ad-pad4 adeno-cmv-padi43*flag-tagged sv40-egfp  (Genechem)

Journal: Cell Communication and Signaling : CCS

Article Title: DNM1L-mediated fission governs mitophagy & mitochondrial biogenesis during myogenic differentiation

doi: 10.1186/s12964-025-02142-x

Figure Lengend Snippet: DNM1L OE induces mitochondrial fragmentation and upregulation of mitophagy during myogenesis. A Representative images of myoblasts transduced with Ad-CMV- Cox8 - EGFP - mCherry to detect mitophagic flux. Mitochondria are dual‐labeled EGFP and mCherry (i.e., yellow). EGFP florescence is reduced during mitochondria degradation resulting in red fluorescence only (i.e., red puncta) and is indicative of mitophagic flux. Scale bar indicates 10 μm. Quantification of ( B ) mitophagic area, ( C ) total mitochondrial branch count, and ( D ) total mitochondrial junction count per cell. Representative histograms and quantification of ( E–F ) MitoTracker Green (MTG) fluorescence, ( G-H ) TMRE fluorescence, and ( I-J ) Calcein-AM + CoCl 2 fluorescence. Quantification of ( K ) CASP9, ( L ) CASP3, and ( M ) CAPN activity at D1. * p < 0.05 compared to Ad- LacZ group within the same time point. n = 4–30 per group

Article Snippet: C2C12 myoblasts were grown on coverslips coated with Cultrex (Trevigen, 3432–010-01) in a 24-well plate and transduced in the same manner with the addition of Ad-CMV- Cox8 - EGFP - mCherry .

Techniques: Transduction, Labeling, Fluorescence, Activity Assay

Journal: Cell Communication and Signaling : CCS

Article Title: DNM1L-mediated fission governs mitophagy & mitochondrial biogenesis during myogenic differentiation

doi: 10.1186/s12964-025-02142-x

Figure Lengend Snippet: DNM1L KD suppressed mitochondrial fission and mitophagy during myogenesis. A Representative images of myoblasts transduced with Ad‐CMV‐ Cox8 ‐ EGFP ‐ mCherry to detect mitophagic flux. Mitochondria are dual‐labeled EGFP and mCherry (i.e., yellow). EGFP florescence is reduced during mitochondria degradation resulting in red fluorescence only (i.e., red puncta) and is indicative of mitophagic flux. Scale bar indicates 10 μm. Quantification of ( B ) mitophagic area, ( C ) total mitochondrial branch count, and ( D ) total mitochondrial junction count per cell. Representative histograms and quantification of ( E–F ) MitoTracker Green (MTG) fluorescence, ( G-H ) TMRE fluorescence, and ( I-J ) Calcein-AM + CoCl 2 fluorescence. Quantification of ( K ) CASP9, ( L ) CASP3, and ( M ) CAPN activity at D1. * p < 0.05 compared to Ad- LacZ group within the same time point. n = 4–30 per group

Article Snippet: C2C12 myoblasts were grown on coverslips coated with Cultrex (Trevigen, 3432–010-01) in a 24-well plate and transduced in the same manner with the addition of Ad-CMV- Cox8 - EGFP - mCherry .

Techniques: Transduction, Labeling, Fluorescence, Activity Assay

Journal: Cell Communication and Signaling : CCS

Article Title: DNM1L-mediated fission governs mitophagy & mitochondrial biogenesis during myogenic differentiation

doi: 10.1186/s12964-025-02142-x

Figure Lengend Snippet: BNIP3 OE activates mitophagy and suppressed CASP activity in fission-deficient cells. A Representative images of myoblasts transduced with Ad-CMV- Cox8 - EGFP - mCherry to detect mitophagic flux. Mitochondria are dual‐labeled EGFP and mCherry (i.e., yellow). EGFP fluorescence is reduced during mitochondria degradation resulting in red fluorescence only (i.e., red puncta) and is indicative of mitophagic flux. Scale bar indicates 10 μm. Quantification of ( B ) mitophagic area, ( C ) total mitochondrial branch count, and ( D ) total mitochondrial junction count per cell. Representative histograms and quantification of ( E–F ) MitoTracker Green (MTG) fluorescence, ( G-H ) TMRE fluorescence, and ( I-J ) Calcein-AM + CoCl 2 fluorescence. Quantification of ( K ) CASP9, ( L ) CASP3, and ( M ) CAPN activity at D1. * p < 0.05 compared to Ad- LacZ group within the same time point. n = 4–30 per group

Article Snippet: C2C12 myoblasts were grown on coverslips coated with Cultrex (Trevigen, 3432–010-01) in a 24-well plate and transduced in the same manner with the addition of Ad-CMV- Cox8 - EGFP - mCherry .

Techniques: Activity Assay, Transduction, Labeling, Fluorescence

Journal: Cell Communication and Signaling : CCS

Article Title: DNM1L-mediated fission governs mitophagy & mitochondrial biogenesis during myogenic differentiation

doi: 10.1186/s12964-025-02142-x

Figure Lengend Snippet: DNM1L OE in Bnip3 −/− cells increased mitochondrial fragmentation and mitophagy. A Representative images of myoblasts transduced with Ad-CMV- Cox8 - EGFP - mCherry to detect mitophagic flux. Mitochondria are dual‐labeled EGFP and mCherry (i.e., yellow). EGFP fluorescence is reduced during mitochondria degradation resulting in red fluorescence only (i.e., red puncta) and is indicative of mitophagic flux. Scale bar indicates 10 μm. Quantification of ( B ) mitophagic area, ( C ) total mitochondrial branch count, and ( D ) total mitochondrial junction count per cell. Representative histograms and quantification of ( E–F ) MitoTracker Green (MTG) fluorescence, ( G-H ) TMRE fluorescence, and ( I-J ) Calcein-AM + CoCl 2 fluorescence. Quantification of ( K ) CASP9, ( L ) CASP3, and ( M ) CAPN activity at D1. * p < 0.05 compared to Ad- LacZ group within the same time point. n = 4–30 per group

Article Snippet: C2C12 myoblasts were grown on coverslips coated with Cultrex (Trevigen, 3432–010-01) in a 24-well plate and transduced in the same manner with the addition of Ad-CMV- Cox8 - EGFP - mCherry .

Techniques: Transduction, Labeling, Fluorescence, Activity Assay

Journal: Redox Biology

Article Title: Inhibition of S100A8/A9 ameliorates neuroinflammation by blocking NET formation following traumatic brain injury ☆

doi: 10.1016/j.redox.2025.103532

Figure Lengend Snippet: PAD4 deficiency abolished the effects of S100A8/A9 on neurological functions and inflammatory/apoptotic protein levels after TBI . A Quantitative analysis of DNA in the supernatant from neutrophils in vitro treated with S100A8/A9. ∗∗p < 0.01, ∗∗∗p < 0.001, n = 6 per group. B, C Representative immunofluorescence staining NETs (H3cit, green) (B) and quantitative analyses of H3cit-positive cells (C) in neutrophils in vitro. Nuclei were stained with DAPI (blue). Scale bar = 50 μm ∗∗∗p < 0.001, n = 6 per group. D, E Representative immunofluorescence staining (D) of DCFH-DA (green) and quantitative analyses of fluorescence intensity of DCFH-DA (E) in neutrophils in vitro. Scale bar = 50 μm ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n = 6 per group. F Quantitative analyses of plasma DNA at 3 d after TBI. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n = 6 per group. G, H mNSS test (G) and Rotarod test (H) at 3 d post-TBI. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n = 8 per group. I–K pro-inflammation-associated mRNA levels were evaluated including CD86 (I) , IL-1β (J) , and IL-6 (K) . ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n = 8 per group. L, M anti-inflammation-associated mRNA levels were evaluated including CD206 (L) and Arginase-1 (M) . ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n = 8 per group. N Representative image of the FJC (green) staining in the cortex at 3 d after TBI. Nuclei were stained with DAPI (blue). Scale bar = 50 μm. O Quantitative analyses of FJC-positive cells in the peri-contusional cortex at 3 d after TBI. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n = 6 per group.

Article Snippet: Recombinant PAD4 adenovirus (Ad-PAD4; Adeno-CMV-Padi4-3∗flag-tagged SV40-EGFP, 4 × 10 10 plaque-forming-unit/mL) and control adenovirus (Ad-con, Adeno-CMV-3∗flag-tagged SV40-EGFP) were purchased from Genechem (Shanghai, China).

Techniques: In Vitro, Immunofluorescence, Staining, Fluorescence, Clinical Proteomics

Journal: Redox Biology

Article Title: Inhibition of S100A8/A9 ameliorates neuroinflammation by blocking NET formation following traumatic brain injury ☆

doi: 10.1016/j.redox.2025.103532

Figure Lengend Snippet: PAD4 overexpression abolished the neuroprotective effects of PAQ on neurological functions and inflammatory/apoptotic proteins levels after TBI . A Schematic image of adeno-PAD4-EGFP and overexpression of PAD4 in the cortex of the mouse. B, C mNSS test (B) and Rotarod test (C) on days 3 post-TBI. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n = 8 per group. D Representative double immunofluorescence staining for Iba1 (red) and CD16/32 (green) in the contused cortex at 3 d after TBI. Nuclei were stained with DAPI (blue). Scale bar = 50 μm. E Quantitative analyses of CD16/32-positive microglia/macrophage in the peri-contusional cortex at 3 d after TBI. ∗∗p < 0.01, ∗∗∗p < 0.001, n = 6 per group. F Representative double immunofluorescence staining for Iba1 (red) and Arginase-1 (green) in the contused cortex at 3 d after TBI. Nuclei were stained with DAPI (blue). Scale bar = 50 μm. G Quantitative analyses of Arginase-positive microglia/macrophage in the peri-contusional cortex at 3 d after TBI. ∗∗∗p < 0.001, n = 6 per group. H Representative image of the FJC (green) staining in the cortex at 3 d after TBI. Nuclei were stained with DAPI (blue). Scale bar = 50 μm. I Quantitative analyses of FJC-positive cells in the peri-contusional cortex at 3 d after TBI. ∗∗∗p < 0.001, n = 6 per group. J Representative image of the TUNEL (green) co-localization with neurons (NeuN, red) in the cortex at 3 d after TBI. Nuclei were stained with DAPI (blue). Scale bar = 50 μm. K Quantitative analyses of TUNEL-positive neurons in the peri-contusional cortex at 3 d after TBI. ∗p < 0.05, ∗∗p < 0.01, n = 6 per group. L Representative Western blot bands and densitometric quantification of TNF-α, IL-1β, Arginase-1 , Bcl-2, Bax , and C-Caspase-3 after TBI. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n = 6 per group.

Article Snippet: Recombinant PAD4 adenovirus (Ad-PAD4; Adeno-CMV-Padi4-3∗flag-tagged SV40-EGFP, 4 × 10 10 plaque-forming-unit/mL) and control adenovirus (Ad-con, Adeno-CMV-3∗flag-tagged SV40-EGFP) were purchased from Genechem (Shanghai, China).

Techniques: Over Expression, Double Immunofluorescence Staining, Staining, TUNEL Assay, Western Blot