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: 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 ) Fluorescence microscopy of Sod2 expression in zebrafish. Scale bars, 150 and 500 μm. ( B ) Confocal microscopy of Park2 and Lc3b in zebrafish cells, focusing on the abdominal cavity near the liver. Scale bars, 5 μm. ( C ) Immunoblot analysis of Sod2, Lc3, and Park2 proteins in zebrafish. ( D ) Transmission electron microscopy of zebrafish cells near the intestine. Label 1: normal mitochondria; label 2: mitophagy process; label 3: lipid droplets; label 4: swollen mitochondria. The captured mitophagy process primarily represents the stage where lysosomes fuse with mitochondrial vesicles or mitochondrial vesicles enclose lysosomes. Scale bars, 1 and 0.5 μm. ( E to G ) Quantitative immunoblot results for Sod2, Lc3, and Park2 proteins in zebrafish. n = 3 independent biological repeats. All data are presented as the means ± SEM. Two-tailed Student’s t test; n.s., not significant; * P < 0.05; ** P < 0.01 compared with the NPC1-KO 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: Fluorescence, Microscopy, Expressing, Confocal Microscopy, Western Blot, Transmission Assay, Electron Microscopy, Two Tailed Test

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

Journal: Bioactive Materials

Article Title: Near infrared enhanced palladium loaded siraitia grosvenorii carbon dots amplify mitophagy for acute lung injury immunotherapy

doi: 10.1016/j.bioactmat.2026.02.040

Figure Lengend Snippet: ALI therapeutic mechanism. A) Co-immunofluorescent staining images of cells incubated with Cy5-CPs or Cy5-CPs@SS31 by confocal staining microscope: DAPI (blue), Mito-tracker green (green), and Cy5-CPs or Cy5-CPs@SS31 (red). (Scale bar = 10 μm) B) MMP of treated cells by flow cytometry. C) Heatmap of DEGs between control group and CPs@SS31+NIR: genes with relatively high (red) and low (blue) expression levels. D) Volcano plot of DEGs: 542 upregulated and 499 downregulated genes between control group and CPs@SS31+NIR. E) Anti-inflammation pathways related DEGs by KEGG enrichment analysis (red). F) Anti-inflammation pathways related differential biological functions by GO enrichment analysis (red): molecular function (MF), biological process (BP) and cell component (CC). G) Protein-protein interaction network of mitophagy related proteins. H) P62, Parkin and PINK1 expression levels of treated cells by WB. I) P62, Parkin and PINK1 expression levels in the lung tissue of treated rats. (Scale bar = 100 μm).

Article Snippet: Furthermore, the membranes were separately incubated with the primary antibody (anti-P62, Parkin, PINK1 and GAPDH, Proteintech, China) overnight at 4 °C.

Techniques: Staining, Incubation, Microscopy, Flow Cytometry, Control, Expressing

Journal: Antioxidants

Article Title: PET117 Deficiency Confers Ferroptosis Resistance Through ACSF2 Downregulation in Cervical Cancer

doi: 10.3390/antiox15070876

Figure Lengend Snippet: Loss of PET117 confers resistance to erastin- and RSL3-induced ferroptosis. ( A ) Control and PET117 -KO HeLa cells were treated with erastin (40 μM), Fer-1 (10 μM) or their combination for 24 h. Cell viability was determined by the CCK-8 assay and analyzed by one-way ANOVA ( n = 6, *: p < 0.05, ***: p < 0.001, n.s., not significant). ( B ) Control and PET117 -KO HeLa cells were treated with RSL3 (4 μM), Fer-1 (10 μM) or their combination for 24 h. Cell viability was determined by the CCK-8 assay (*: p < 0.05, ***: p < 0.001, n.s., not significant). ( C ) Cell morphology was observed under an inverted light microscope. Scale bar = 50 µm. ( D , E ) Relative cell viability of control, PET117 -KO and PET117 -KO HeLa cells expressing exogenous PET117 were treated with erastin ( D ) or RSL3 ( E ) ( n = 4, ***: p < 0.001, n.s). ( F , G ) Immunoblotting assays of Caspase3, PARP, and BCL-2 in the control and PET117 -KO HeLa cells. β-ACTIN was used as loading controls. Quantified intensities were presented in bar graph ( G ) as means ± s.e.m. and analyzed by a two-tailed paired Student’s t -test ( n = 3, n.s., not significant). ( H , I ) Immunoblotting assays of p62, Parkin, and in the control and PET117 -KO HeLa cells. β-ACTIN was used as loading controls. Quantified intensities were presented in bar graph ( I ) as means ± s.e.m. and analyzed by a two-tailed paired Student’s t -test ( n = 3, n.s., not significant).

Article Snippet: The following primary antibodies were used in this study: PET117 (Invitrogen, PA5-61574, Carlsbad, CA, USA), β-ACTIN (Cell Signaling Technology, #3700S, Danvers, MA, USA), α-TUBULIN (Cell Signaling Technology, #3873S, Danvers, MA, USA), SDHB (Santa cruz, #271548, Santa Cruz, CA, USA), Histone H3 (Abcam, #ab1791, Cambridge, UK), Caspase3 (Cell Signaling Technology, #14220S, Danvers, MA, USA), PARP (Cell Signaling Technology, #9532S, Danvers, MA, USA), BCL-2 (Cell Signaling Technology, #3498S, Danvers, MA, USA), LC3B (Cell Signaling Technology, #3868, Danvers, MA, USA), P62 (Abclonal, #A19700, Wuhan, China), Parkin (Abclonal, #A0968), ACSF2 (Proteintech, # 16140-1-AP, Wuhan, China), and GPX4 (Proteintech, # 30388-1-AP, Wuhan, China).

Techniques: Control, CCK-8 Assay, Light Microscopy, Expressing, Western Blot, Two Tailed Test