Journal: PLOS One

Article Title: Mitochondria-targeted ROS scavenger JP4-039 improves cardiac function in a post-myocardial infarction animal model and induces angiogenesis in vitro

doi: 10.1371/journal.pone.0320703

Figure Lengend Snippet: In acute MI, the exacerbation of Mito-ROS leads to mitochondrial dysfunction and a decrease in ATP production, leading to an increase in AMP/ATP ratio, which results in phosphorylation of AMPKα at Threonine 172. Over time, the unmet need for ATP results in impaired angiogenesis and loss of capillaries in the heart, affecting blood supply and myocardial healing. This leads to cardiac maladaptation, resulting in impaired cardiac function associated with larger infarcted tissue area after acute MI. When JP4-039 is introduced after acute MI, its mitochondria-targeted ROS and electron scavenging activities result in a reduction of Mito-ROS and increased expression of mitochondrial complexes I and V. Thus, treatment with JP4-039 results in increased ATP supply and reduced phosphorylation of AMPKα at Threonine 172 in comparison to untreated mice after MI, suggesting an improvement of the AMP/ATP balance. The increase in ATP supply promoted by JP4-039 may contribute to angiogenesis associated with an improved cardiac function and reduction of infarct size after an acute MI. Created with BioRender.com.

Article Snippet: Human coronary artery endothelial cells (HCAEC), or MHEC with prolonged overexpression of NOX2 were plated into a 15-well µ-slide Angiogenesis (ibidi GmbH, Gräfelfing, Germany) precoated with Cultrex® basement membrane extract (BME, R&D Systems, Minneapolis, MN) at a density of 10 4 cells/well (passage 4), following manufacturer protocol [ ].

Techniques: Expressing, Comparison

Journal: Molecular Therapy

Article Title: The role of the mitochondrial protein VDAC1 in inflammatory bowel disease: a potential therapeutic target

doi: 10.1016/j.ymthe.2021.06.024

Figure Lengend Snippet: VDAC1 is overexpressed in human colon pathologies and in DSS-treated cells, which result in VDAC1 oligomerization, apoptosis, and mtDNA release that are inhibited by VBIT-12 Immunohistochemical (IHC) staining of VDAC1 was performed on tissue microarray slides obtained from Biomax US (Cat No. CO245). The array contains colon sections from healthy (H, 4 samples), colitis (Co, 4 samples), ulcerative colitis (UC, 4 samples), and Crohn’s disease of the ileocecal junction (CD, 4 samples). (A) Representative sections of the indicated tissues were IHC stained for VDAC1 using specific antibodies. Scale bars are 20 or 50 μm, as indicated. (B) Quantitation of VDAC1 expression levels in the whole area of the provided sections. Staining intensity was quantified using a panoramic microscope and HistoQuant software. Results, mean ± SEM, ∗∗p < 0.01, ∗∗∗p < 0.001, NS not significant (n = 4 for each group). (C–E) Representative images of colon tissue sections from control and DSS-induced colitis mice, IHC, stained for VDAC1 (C; n = 6 for control and n = 7 for DSS). Scale bar is 50 μm. (D–I) CT-26 cells were incubated with the indicated DSS concentration for 48 h in the presence or absence of VBIT-12 (10 or 20 μM) (n = 3). Then samples were analyzed for VDAC1 expression level by immunoblotting with acting serving as loading control (D), oligomerization (E), apoptosis (assayed by PI staining and flow cytometry) (F), intracellular Ca 2+ levels (G), mtROS (H), and mtDNA release (I); all were assayed as described in the Materials and methods section. Results, mean ± SEM, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, n = 3, NS, not significant. (J) Proposed coupling of VDAC1 overexpression induced by pathological conditions (IBD) and VDAC1 oligomerization mediating the release of apoptogenic proteins from the IMS, leading to apoptosis and the release of mtDNA, leading to inflammasome activation. These processes are inhibited by the VDAC1-interacting molecules, VBIT-4 and VBIT-12, via preventing VDAC1 oligomerization.

Article Snippet: VDAC1 is overexpressed in human colon pathologies and in DSS-treated cells, which result in VDAC1 oligomerization, apoptosis, and mtDNA release that are inhibited by VBIT-12 Immunohistochemical (IHC) staining of VDAC1 was performed on tissue microarray slides obtained from Biomax US (Cat No. CO245).

Techniques: Immunohistochemical staining, Immunohistochemistry, Microarray, Staining, Quantitation Assay, Expressing, Microscopy, Software, Control, Incubation, Concentration Assay, Western Blot, Flow Cytometry, Over Expression, Activation Assay

Journal: bioRxiv

Article Title: Targeting NEDD9-SH3 with a Covalent Peptide Controls Endothelial Phenotype

doi: 10.1101/2025.07.10.663547

Figure Lengend Snippet: a. Paraffin-embedded lung sections from control, rat SU-5416-Hypoxia-Normoxia-PAH, and rat monocrotaline (MCT)-PAH ( n =4-5 rats/condition) or isolated from idiopathic PAH patients ( n =5-8/condition) at the time of lung explant were stained using anti-PECAM1 and anti-NRP-1 antibodies and the immunohistochemical profile in distal pulmonary arterioles was analyzed. Scale bar, 20 μm. b. Human pulmonary artery endothelial cells (HPAECs) and human pulmonary artery smooth muscle cells were co-cultured in a barrier-free system to recapitulate the direct cell-cell contact conditions in PAH lesions. Double immunogold-labeled staining was performed using anti-NRP-1 and anti-Heparan sulfate antibodies, and imaged using transmission electron microscopy. Scale bar, 500 nm. Inset scale bar, 100 nm. c. Disease prevention protocol using the rat SU-5416-hypoxia-normoxia and monocrotaline (MCT) experimental models of PAH and timepoints for intratracheal administration of si-Scrambled (negative) control (Scr) or siRNA against NEDD9. d. The right ventricular systolic pressure (RVSP) measured by cardiac catheterization ( n =3-7 rats/condition) and e. RV mass measured by Fulton Index. IVs, interventricular septum; LV, left ventricle. f. Human pulmonary artery endothelial cells were untreated or treated with VEGF-C (100 nM) for 6 hr and transfected with vehicle (V) control or adenovirus (Ad) containing green fluorescent protein (GFP), Sulf1 cDNA, or siRNA-Sulf1, and cell migration was quantitated using the wound healing assay. Black line, separate experiment iteration without VEGF-C or other treatment ( n =4-7). Blue, DAPI. Scale bar, 400 μm. g. HPAECs were untransfected or transfected with an Ad carrying vehicle (V) control, Scr, Sulf1, or si-Sulf1 and stained with an anti-FAK-Y861 antibody. The number of podosome rosette-positive cells (podosome+) per low power field (40x) and the number of cells with >2 podosomes was quantitated ( n =3). Arrows indicate podosome structures. Scale bar, 50 μm. h. The number of tubes formed per 10x field was counted for untreated HPAECs and cells transfected with Ad carrying V, Sulf1, and si-Sulf1 ( n =3). Scale bar, 300 μm. i . The effect of Ad-Sulf1 on collagen III deposition in HPAECs ( n =5-10). PFE, plaque forming unit (N=1-3 PFEs). Scale bar, 50 μm.

Article Snippet: To analyze the effect of Sulf1 overexpression or inhibition on tube formation, HPAECs were incubated for 24 hours with adenovirus expressing GFP (Ad-GFP), GFP plus human Sulfatase-1 (Ad-Sulf1) or GFP plus human Sulf1-siRNA (Ad-siSulf1), 1 PFU/ml x10 7 (Vector Biolabs) before plating on extracellular matrix-coated slides.

Techniques: Control, Isolation, Staining, Immunohistochemical staining, Cell Culture, Labeling, Transmission Assay, Electron Microscopy, Negative Control, Transfection, Migration, Wound Healing Assay

Journal: Frontiers in Cell and Developmental Biology

Article Title: LncRNA FEZF1-AS1 Promotes Multi-Drug Resistance of Gastric Cancer Cells via Upregulating ATG5

doi: 10.3389/fcell.2021.749129

Figure Lengend Snippet: ATG5 knockdown inhibited autophagy of GC cells promoted by overexpression of LncFEZF1-AS1. (A) CCK8 assay on the proliferation of SGC-7901 cells with or without the treatment of FEZF1-AS1, FEZF1-AS1 + ATG5 siRNA or their control. (B) The proliferation of SGC-7901 with or without the treatment of FEZF1-AS1 siRNA, FEZF1-AS1 siRNA + ATG5 or their control group analyzed by CCK8 assay. (C) Representative images of LC3 puncta formation in SGC-7901 observed via IF staining, green: LC3B, bule: DAPI, scar bar: 20 mM. The results represented the mean ± SD of 3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Cells were cultured on 24-well chamber slides and co-transfected with FEZF1-AS1 overexpression plasmids and si-ATG5 for 48 h. At the time of harvest, cells were washed with PBS and fixed with 4% paraformaldehyde for 15 min and then permeabilized with 0.01% Triton X-100 for 30 min. Then cells were stained with anti-LC3B antibody (Proteintech) overnight at 4°C.

Techniques: Knockdown, Over Expression, CCK-8 Assay, Control, Staining