Journal: Journal of Cardiovascular Development and Disease

Article Title: Endothelial Filamin C Alleviates Atherosclerosis via PINK1/Parkin-Dependent Mitophagy and mtDNA-cGAS-STING Inflammation Suppression

doi: 10.3390/jcdd13090428

Figure Lengend Snippet: FLNC activates PINK1/Parkin-mediated mitophagy. Stable HUVEC lines with FLNC overexpression (OE) or knockdown (KD) were established using CRISPRa and CRISPRi systems, respectively, and the effect of FLNC on PINK1/Parkin-mediated mitophagy was evaluated. ( A ) Transmission electron microscopy (TEM) analysis of mitochondrial ultrastructure and autophagic flux. Representative images are shown. Black arrows: normal mitochondria; green arrows: endoplasmic reticulum; red arrows: autophagosomes; blue arrows: autolysosomes; yellow arrows: damaged mitochondria (scale bar: 1 μm). ( B ) Western blotting analysis of mitophagy-related proteins (PINK1, Parkin, LC3II, p62, and TOM20) (n = 4). ( C , D ) Co-immunofluorescence staining of TOM20 with PINK1 or Parkin (scale bar: 17 μm, n = 4). ( E , F ) Co-immunofluorescence staining of TOM20 with LC3 (autophagosome marker) or LAMP1 (lysosome marker) (scale bar: 17 μm, n = 4). ( G ) Mitochondrial ROS (mtROS) levels were measured by MitoSOX staining (scale bar: 200 μm, n = 4). ( H ) JC-1 fluorescence labeling was applied to assess mitochondrial membrane potential (scale bar: 200 μm, n = 4).

Article Snippet: Incubation steps were performed on sections using single primary antibodies or multiplex primary antibody combinations during overnight incubation at 4 °C: TOM20 (1:200, A27799, ABclonal)/PINK1 (1:200, A7131, ABclonal), TOM20 (1:200, A27799, ABclonal)/Parkin (1:200, A0968, ABclonal), TOM20 (1:200, A27799, ABclonal)/LC3 (1:200, A19665, ABclonal), TOM20 (1:200, AF1717, Beyotime)/LAMP1 (1:200, SC20011, Santa Cruz, Singapore), TOM20 (1:200, A27799, ABclonal)/TFAM (1:200, A23467, ABclonal), cleaved-caspase-3 (1:500, 68773-1-Ig, Proteintech), CD31 (1:800, 66065-2-Ig, Proteintech)/FLNC (1:200, A13018, ABclonal), CD31 (1:200, 28083-1-AP, Proteintech)/cleaved-caspase-3 (1:500, 68773-1-Ig, Proteintech), α-SMA(1:200, 67735-1-Ig, Proteintech)/FLNC(1:200, A13018, ABclonal).

Techniques: Over Expression, Knockdown, Transmission Assay, Electron Microscopy, Analysis, Western Blot, Immunofluorescence, Staining, Marker, Fluorescence, Labeling, Membrane

Journal: Journal of Cardiovascular Development and Disease

Article Title: Endothelial Filamin C Alleviates Atherosclerosis via PINK1/Parkin-Dependent Mitophagy and mtDNA-cGAS-STING Inflammation Suppression

doi: 10.3390/jcdd13090428

Figure Lengend Snippet: FLNC suppresses mtDNA release and inhibits the cGAS-STING inflammatory pathway. The effect of FLNC on mitochondrial DNA leakage and cGAS-STING signaling cascade induction was investigated. ( A ) Co-immunofluorescence staining of TOM20 (mitochondrial marker, green) and TFAM (mtDNA-binding protein, red). Red arrows indicate TFAM-positive regions not co-localized with TOM20 (scale bar: 8 μm). ( B ) Subcellular fractionation followed by Western blotting was applied for the measurement of TFAM levels across cytoplasmic and mitochondrial compartments (n = 4). The purity of the isolated fractions was confirmed by the absence of TOM20 in cytoplasmic extracts and β-actin in mitochondrial preparations. ( C ) PicoGreen fluorescence staining of cytosolic double-stranded DNA in HUVECs (n = 4) (scale bar: 100 μm). ( D ) Western blotting to profile key mediators of cGAS-STING inflammatory axis components and downstream pro-inflammatory cytokines (n = 4). ( E ) Western blotting quantifying MCP-1 protein levels within human carotid plaques and corresponding non-lesioned perilesional vasculature (n = 20). Phosphorylated and total protein levels were detected on the same blots after membrane stripping with Western Stripping buffer.

Article Snippet: Incubation steps were performed on sections using single primary antibodies or multiplex primary antibody combinations during overnight incubation at 4 °C: TOM20 (1:200, A27799, ABclonal)/PINK1 (1:200, A7131, ABclonal), TOM20 (1:200, A27799, ABclonal)/Parkin (1:200, A0968, ABclonal), TOM20 (1:200, A27799, ABclonal)/LC3 (1:200, A19665, ABclonal), TOM20 (1:200, AF1717, Beyotime)/LAMP1 (1:200, SC20011, Santa Cruz, Singapore), TOM20 (1:200, A27799, ABclonal)/TFAM (1:200, A23467, ABclonal), cleaved-caspase-3 (1:500, 68773-1-Ig, Proteintech), CD31 (1:800, 66065-2-Ig, Proteintech)/FLNC (1:200, A13018, ABclonal), CD31 (1:200, 28083-1-AP, Proteintech)/cleaved-caspase-3 (1:500, 68773-1-Ig, Proteintech), α-SMA(1:200, 67735-1-Ig, Proteintech)/FLNC(1:200, A13018, ABclonal).

Techniques: Immunofluorescence, Staining, Marker, Binding Assay, Cell Fractionation, Western Blot, Isolation, Fluorescence, Membrane, Stripping

Journal: Journal of Cardiovascular Development and Disease

Article Title: Endothelial Filamin C Alleviates Atherosclerosis via PINK1/Parkin-Dependent Mitophagy and mtDNA-cGAS-STING Inflammation Suppression

doi: 10.3390/jcdd13090428

Figure Lengend Snippet: FLNC exerts its protective effects in a mitophagy-dependent manner. Mdivi-1, a pharmacologic blocker of mitophagy, was utilized for rescue experiments. ( A ) Western blotting analysis of mitophagy-related proteins and cleaved-caspase-3. PINK1 and Parkin (n = 4); LC3II, p62, TOM20, and cleaved-caspase-3 (n = 8). ( B ) Immunofluorescent labeling of cell coverslips targeting cleaved caspase-3 (scale bar: 100 μm, n = 4). ( C ) TUNEL assay was used to detect endothelial cell apoptosis (scale bar: 100 μm, n = 4). ( D ) Intracellular ROS levels were measured by DCFH-DA staining (scale bar: 200 μm, n = 4). ( E ) JC-1 fluorochrome was utilized for the quantification of mitochondrial membrane potential (scale bar: 100 μm, n = 4). ( F ) Western blotting analysis of cGAS-STING inflammatory signaling axis components and downstream pro-inflammatory cytokines (n = 4). Phosphorylated and total protein levels were detected on the same blots after membrane stripping with Western Stripping buffer.

Article Snippet: Incubation steps were performed on sections using single primary antibodies or multiplex primary antibody combinations during overnight incubation at 4 °C: TOM20 (1:200, A27799, ABclonal)/PINK1 (1:200, A7131, ABclonal), TOM20 (1:200, A27799, ABclonal)/Parkin (1:200, A0968, ABclonal), TOM20 (1:200, A27799, ABclonal)/LC3 (1:200, A19665, ABclonal), TOM20 (1:200, AF1717, Beyotime)/LAMP1 (1:200, SC20011, Santa Cruz, Singapore), TOM20 (1:200, A27799, ABclonal)/TFAM (1:200, A23467, ABclonal), cleaved-caspase-3 (1:500, 68773-1-Ig, Proteintech), CD31 (1:800, 66065-2-Ig, Proteintech)/FLNC (1:200, A13018, ABclonal), CD31 (1:200, 28083-1-AP, Proteintech)/cleaved-caspase-3 (1:500, 68773-1-Ig, Proteintech), α-SMA(1:200, 67735-1-Ig, Proteintech)/FLNC(1:200, A13018, ABclonal).

Techniques: Western Blot, Analysis, Labeling, TUNEL Assay, Staining, Membrane, Stripping

Journal: Journal of Cardiovascular Development and Disease

Article Title: Endothelial Filamin C Alleviates Atherosclerosis via PINK1/Parkin-Dependent Mitophagy and mtDNA-cGAS-STING Inflammation Suppression

doi: 10.3390/jcdd13090428

Figure Lengend Snippet: FLNC exerts its protective effects via the PINK1/Parkin signaling pathway. Rescue experiments were performed by transfecting cells with siRNAs targeting PINK1 or Parkin. ( A ) Western blotting analysis of cleaved-caspase-3 expression (n = 4). ( B ) Immunofluorescence staining targeting cleaved-caspase-3 (scale bar: 100 μm, n = 4). ( C ) TUNEL assay was used to detect endothelial cell apoptosis (scale bar: 100 μm, n = 4). ( D ) DCFH-DA staining was applied to quantify intracellular reactive oxygen species (scale bar: 200 μm, n = 4). ( E ) Western blotting analysis of mitophagy-related proteins (n = 4). ( F , G ) Dual immunofluorescence staining was performed for TOM20 paired with PINK1, and TOM20 paired with Parkin separately; co-localization was quantified using Pearson correlation coefficient (scale bar: 17 μm, n = 4). ( H , I ) Co-immunofluorescence staining of TOM20 with LC3 (autophagosome marker) and LAMP1 (lysosome marker); co-localization was quantified using Manders overlap coefficient (scale bar: 17 μm, n = 4). ( J ) TEM analysis of mitochondrial ultrastructure and autophagic flux. Black arrows: normal mitochondria; red arrows: autophagosomes; blue arrows: autolysosomes; yellow arrows: damaged mitochondria (scale bar: 1 μm). ( K ) Co-immunofluorescence staining of TOM20 and TFAM (scale bar: 8 μm). ( L ) Subcellular fractionation followed by Western blotting was performed to detect cytoplasmic TFAM levels (n = 4). The purity of the isolated fractions was confirmed by the absence of TOM20 in cytoplasmic extracts and β-actin in mitochondrial preparations. ( M ) PicoGreen fluorescence staining of cytosolic double-stranded DNA in HUVECs (n = 4) (scale bar: 100 μm). ( N ) Protein levels of cGAS-STING axis members as well as downstream pro-inflammatory mediators were determined via Western blotting (n = 4). Phosphorylated and total protein levels were detected on the same blots after membrane stripping with Western Stripping buffer.

Article Snippet: Incubation steps were performed on sections using single primary antibodies or multiplex primary antibody combinations during overnight incubation at 4 °C: TOM20 (1:200, A27799, ABclonal)/PINK1 (1:200, A7131, ABclonal), TOM20 (1:200, A27799, ABclonal)/Parkin (1:200, A0968, ABclonal), TOM20 (1:200, A27799, ABclonal)/LC3 (1:200, A19665, ABclonal), TOM20 (1:200, AF1717, Beyotime)/LAMP1 (1:200, SC20011, Santa Cruz, Singapore), TOM20 (1:200, A27799, ABclonal)/TFAM (1:200, A23467, ABclonal), cleaved-caspase-3 (1:500, 68773-1-Ig, Proteintech), CD31 (1:800, 66065-2-Ig, Proteintech)/FLNC (1:200, A13018, ABclonal), CD31 (1:200, 28083-1-AP, Proteintech)/cleaved-caspase-3 (1:500, 68773-1-Ig, Proteintech), α-SMA(1:200, 67735-1-Ig, Proteintech)/FLNC(1:200, A13018, ABclonal).

Techniques: Western Blot, Analysis, Expressing, Immunofluorescence, Staining, TUNEL Assay, Marker, Cell Fractionation, Isolation, Fluorescence, Membrane, Stripping

Journal: Antioxidants

Article Title: Calycosin Safeguards Mitochondrial Genomic Stability and Iron Homeostasis via the NRF2/NRF1-TFAM Axis to Mitigate Doxorubicin-Induced Cardiotoxicity

doi: 10.3390/antiox15091058

Figure Lengend Snippet: Calycosin ameliorates DOX-mediated mitochondrial dysfunction in vivo. ( A ) TEM analysis showing calycosin treatment preserved mitochondrial architecture ( n = 3 mice per group). ( B ) Quantitative analysis of mitochondrial cristae volume density in TEM images ( n = 3 mice per group). ( C , D ) Cardiac oxidative stress parameters (lipid peroxidation marker MDA and antioxidant enzyme SOD; n = 3 mice per group). ( E ) Quantitative colocalization analysis of 8-OHdG and Tom20 using Pearson’s correlation coefficient ( n = 3 mice per group). ( F ) Representative immunofluorescence images illustrating DNA damage evaluated by 8-OHdG staining (scale bar = 20 μm; n = 3 mice per group). Data are presented as mean ± SD. * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 compared with DOX group.

Article Snippet: Immunofluorescence staining was performed using an anti-NRF2 antibody (A21508, ABclonal, Wuhan, China), an anti-8-OHdG antibody (sc-393871, Santa Cruz Biotechnology, Dallas, TX, USA) and an anti-Tom20 antibody (A19403, ABclonal, Wuhan, China) on tissue sections.

Techniques: In Vivo, Analysis, Marker, Immunofluorescence, Staining

Journal: Science Advances

Article Title: Mito-TEMPO improves survival rates in npc1 -knockout zebrafish by reducing oxidative stress and enhancing mitophagy via Sod2

doi: 10.1126/sciadv.aee0509

Figure Lengend Snippet: ( A to F ) Flow cytometry histograms showing LC3B, p62, TOMM20, COX-IV, PINK1, and PARK2 expression in HepG2 cells. For each assay, a blank group was set without antibody but with FITC dye. ( G to L ) Statistical results for LC3B, SQSTM1/p62, TOMM20, COX-IV, PINK1, and PARK2 expression in HepG2 cells. n = 3 independent biological repeats. All data are presented as the means ± SEM. Two-tailed Student’s t test [(G) to (L)]; n.s., not significant; * P < 0.05; ** P < 0.01; *** P < 0.001 compared with the model group.

Article Snippet: Cells were incubated with rabbit anti-mouse primary antibodies against LC3B (1:100, ABclonal, A11923), SQSTM1/p62 (1:100, ABclonal, A11483), PINK1 (1:100, ABclonal, A7131), PARK2 (1:100, ABclonal, A0968), TOMM20 (1:100, ABclonal, A6774), COX-IV (1:100, ABclonal, HB, A6564), or SOD2 (1:100, GeneTex, GTX124294) for 4 hours, followed by FITC-conjugated goat anti-rabbit IgG (1:100, Thermo Fisher Scientific, F-2765) for 1 hour at RT.

Techniques: Flow Cytometry, Expressing, Two Tailed Test