Journal: BMC Complementary Medicine and Therapies

Article Title: Ammidin ameliorates myocardial hypoxia/reoxygenation injury by inhibiting the ACSL4/AMPK/mTOR-mediated ferroptosis pathway

doi: 10.1186/s12906-023-04289-x

Figure Lengend Snippet: Effect of 2 μM Fer-1 on ROS levels in H/R-induced cardiomyocytes. ( A ) Cardiomyocyte viability. ( B ) LDH release. ( C ) MDA content. ( D ) ROS fluorescence image, Merge: A combination of ROS (green fluorescence) and nucleus (blue fluorescence). ( E ) ROS quantitative analysis. ( F ) SOD content. ( G ) JC-1 staining showed that JC-1 aggregated in the mitochondria of normal cells emitted red fluorescence, while JC-1 in dead cells emitted green fluorescence due to its presence in the cytoplasm as a monomer. Merge: A combination of aggregates (red fluorescence), monomers (green fluorescence) and nucleus (blue fluorescence). ( H ) Red/green fluorescence ratio at the MMP level. ( I-K ) Western blotting revealing the expression levels of ACSL4 and GPX4 in cardiomyocytes ( * P < 0.05 vs. control; # P < 0.05 vs. H/R; scale bars, 100 μm; n = 3). Proteins were then transferred onto PVDF membranes. After cutting according to the molecular size, the primary antibody was incubated. Fer-1, ferrostatin-1; ROS, reactive oxygen species; H/R, hypoxia/reoxygenation; LDH, lactate dehydrogenase; MDA, malondialdehyde; SOD, superoxide dismutase; JC-1, 1,1′,3,3′-tetraethyl-5,5′,6,6′-tetrachloroimidacarbocyanine iodide; MMP, mitochondrial membrane potential; ACSL4, Acyl-CoA synthetase long chain family member 4; GPX4, glutathione peroxidase 4

Article Snippet: Anti-β-actin (cat. no. sc-47,778; dilution, 1:1000) and goat anti-rabbit IgG (cat. no. sc-2004; dilution, 1:20,000) were purchased from Santa Cruz Biotechnology, Inc. Primary antibodies against proteins, including ACSL4 (cat. no. ab155282; dilution, 1:1000), GPX4 (cat. no. ab125066; dilution, 1:1000), AMPK (cat. no. ab32047; dilution, 1:1000), phosphorylated (p-AMPK) (cat. no. ab133448; dilution, 1:1000), mTOR (cat. no. ab134903; dilution, 1:500) and p-mTOR (cat. no. ab109 268; dilution, 1:500), were purchased from Abcam.

Techniques: Fluorescence, Staining, Western Blot, Expressing, Control, Incubation, Membrane

Journal: Annals of Translational Medicine

Article Title: Trimethylamine-N-oxide-stimulated hepatocyte-derived exosomes promote inflammation and endothelial dysfunction through nuclear factor-kappa B signaling

doi: 10.21037/atm-21-5043

Figure Lengend Snippet: Isolation and characterization of Exos isolated from hepatocytes culture supernatant. (A) Nanovesicles with diameters of around 100 nm were isolated and purified from the hepatocyte culture supernatant, and visualized under an electron microscope. (B) The size distribution of these nanovesicles were further identified by nanoparticle tracking analysis. (C) The expressions of exosomal identity marker CD81 and TSG101 were detected in the Control-Exos and TMAO-Exos by western blotting. (D) The Exos were labelled with DiI and co-cultured with HAECs for 24 h. The results showed that the DiI-labelled Exos could be taken up by HAECs (400× magnification). Exos, exosomes; CD81, cluster of differentiation 81; TSG101, tumor susceptibility gene 101; TMAO, trimethylamine-N-oxide; HAECs, human aortic endothelial cells.

Article Snippet: CD81 primary antibody was purchased from Servicebio (Wuhan, China), TSG101 was purchased from ZEN BIO (Chengdu, China), nuclear factor-kappa B (NF-κB) p65 and Phospho-NF-κB p65 (p-NF-κB p65) at serine536 (Ser536) were purchased from Cell Signaling Technology (Danvers, MA, USA), and glyceraldehyde phosphate dehydrogenase (GAPDH) was purchased from Proteintech Group (Rosemont, IL, USA).

Techniques: Isolation, Purification, Microscopy, Marker, Control, Western Blot, Cell Culture

Journal: The Journal of Cell Biology

Article Title: Caveolin-1 is ubiquitinated and targeted to intralumenal vesicles in endolysosomes for degradation

doi: 10.1083/jcb.201003086

Figure Lengend Snippet: CAV1 ubiquitination and ESCRT machinery are needed for ILV targeting. (A) In CV1 cells expressing mEGFP-mCherry (tandem)–tagged CAV1, mCherry fluorescence did not overlap with mEGFP fluorescence in many endosomal structures, indicating acid-dependent quenching of mEGFP in LE/LYS . (B and C) Treatment of cells with 20 mM NH 4 Cl (B) or 10 µM MG132 (C) restored mEGFP fluorescence, which is consistent with neutralized lumenal pH and defects in ILV targeting, respectively. HeLa cells pretreated for 48 h with control siRNA (D) or siRNAs targeting ESCRT components Hrs and Tsg101 (E; double knockdown) were transfected with CAV1-tandem for 12 h and viewed live by confocal microscopy. The presence of yellow endosomes upon knockdown of Hrs and Tsg101 showed involvement of ESCRT machinery in ILV targeting of CAV1. (F) Western blot analysis indicated successful knockdown of Hrs and Tsg101. (A–E) Single confocal sections of live cells are shown. (bottom) Insets show enlargements of boxed areas. Bars, 10 µm.

Article Snippet: Rabbit pAb anti-CAV1 (N20; sc-894), mouse mAb anti-LAMP1 (sc-20011), and mouse mAb anti–c-myc (9E10; sc-40) were obtained from Santa Cruz Biotechnology, Inc., rabbit pAb anti-PTRF (polymerase I and transcript release factor; cavin-1) from Abcam (ab48824), rabbit pAb anti-giantin (PRB-114C) and mAb anti-HA from Covance (MMS-101P), mAb anti-Hrs from Sigma-Aldrich (6D11; WH0009146M1), mAb anti-Tsg101 from Axxora (4A10; NB200-112), mouse mAb anti–β-actin from Sigma-Aldrich (AC-15; A1978), and mouse mAb anti-ubiquitin from Enzo Life Sciences, Inc. (FK2; BML-PW8810-0500).

Techniques: Expressing, Fluorescence, Transfection, Confocal Microscopy, Western Blot

Journal: Frontiers in Immunology

Article Title: Downregulated Serum Exosomal miR-451a Expression Correlates With Renal Damage and Its Intercellular Communication Role in Systemic Lupus Erythematosus

doi: 10.3389/fimmu.2021.630112

Figure Lengend Snippet: Validation and function of exosomes extracted from serum samples. (A) Transmission electron microscopy ( n = 3) revealed the spherical characteristics and size (30–110 nm) of exosomes by ultracentrifugation pellet from serum. (B) Western blotting ( n = 3) showed that the pellet after ultracentrifugation was strongly reactive for antibodies against the exosomal membrane markers CD63 and TSG101. Reverse transcription-quantitative PCR ( n = 3) was employed to detect the mRNA expression levels of (C) TNF-α, (D) IFN-γ, and (E) IL-6 in CD4 + T cells co-cultured with exosomes of normal controls(NC) or patients with systemic lupus erythematosus(SLE) for 24 h, The mRNA expression levels of TNF-α, IFN-γ, and IL-6 were increased significantly after treated with SLE-exo while no significant changes in NC-exo group. RT-qPCR results were normalized to GAPDH. * P < 0.05, ** P < 0.01, NS, no significance.

Article Snippet: The membranes were incubated with polyclonal anti-human TSG101 and CD63 antibodies (Bioworld Technology, Inc.) and then with goat anti-rabbit IgG conjugated to Sepharose beads (Bioworld Technology, Inc.).

Techniques: Transmission Assay, Electron Microscopy, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Cell Culture, Quantitative RT-PCR