Journal: Journal of Extracellular Biology

Article Title: TDO2‐augmented fibroblasts secrete EVs enriched in immunomodulatory Y‐derived small RNA

doi: 10.1002/jex2.73

Figure Lengend Snippet: NT4 is a short non‐coding RNA fragment with immunomodulatory function derived from human Y RNA‐4 (hY4). (a) Schematic of the hY4 sequence and functional regions. (b) Sequence of NT4 and its position within the hY4 fragment EV‐YF1. (c) qPCR showing NT4 enrichment in CCM of fibroblasts transduced with TDO2‐activating lentivirus (TDO2‐CCM) compared to those from fibroblasts transduced with a lentivirus containing a control vector (VC‐CCM). (d) QPCR of NT4 from SEC‐isolated EVs compared to the EV‐depleted fraction. (e) Absolute quantification (NT4 copies/10 3 particles) of TDO2‐CCM, VC‐CCM, SEC‐isolated TDO2‐EVs and the EV‐depleted fraction. (f) RNAse protection assay demonstrating NT4 is encapsulated inside TDO2‐EVs. (g) Broad expression of the monocyte marker, CD68, in bone marrow‐derived macrophages. (h) Equivalent uptake of TDO2‐ and HDF‐EVs (EV ceramide dye; red, phalloidin: green and nuclei: blue) by bone marrow derived macrophages at 30, 60 and 180 min post exposure as measured by internalisation of red signals inside macrophages and, (i) area under the curve measurement of cumulative signal at all three time points. (j) Representative image of TDO2‐ and HDF‐EVs (EV ceramide dye; red, phalloidin: green and nuclei: blue) by bone marrow derived macrophages at 1‐h post‐exposure. (k–m) TDO2‐EV and NT4 pre‐treatment lowered expression of inflammatory genes il1b and il6 , as well as pro‐senescence p21 / cdkn1a . Suppression of NT4 in TDO2‐CCM abrogated the suppressive effect of TDO2‐CCM on il1b , il6 and p21/cdkn1a in LPS‐stimulated macrophages. Scale bars: 100 μm. Statistical analysis between two groups was done using Student's T test and comparisons between three or more groups was done using one‐way ANOVA with Sidak's multiple comparison test. Error bars represent standard deviation,* p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: RNA was isolated using the urine EV RNA isolation kit (Norgen Biotek, cat# 47200). qPCR was performed to assess NT4 expression levels.

Techniques: Derivative Assay, Sequencing, Functional Assay, Transduction, Control, Plasmid Preparation, Isolation, Quantitative Proteomics, Rnase Protection Assay, Expressing, Marker, Comparison, Standard Deviation

Journal: Biomedicines

Article Title: Circulating Exosomal miRNAs as Biomarkers in Epithelial Ovarian Cancer

doi: 10.3390/biomedicines9101433

Figure Lengend Snippet: Circulating exosomal miRNAs identified as potential diagnostic biomarkers in EOCs. N/A: not available, HGSC: high-grade serous carcinoma.

Article Snippet: Exosomes were further isolated using differential ultracentrifugation (UC) or by commercial exosomal miRNA isolation kits (Norgen Biotek), and miRNAs were analyzed by next generation sequencing (NGS).

Techniques: Diagnostic Assay, Expressing, TaqMan Assay, Clinical Proteomics

Journal: Biomedicines

Article Title: Circulating Exosomal miRNAs as Biomarkers in Epithelial Ovarian Cancer

doi: 10.3390/biomedicines9101433

Figure Lengend Snippet: Sensitivity, specificity and area under the receiver operating characteristic (ROC) curve (AUC) of the circulating exosomal miRNAs used to detect epithelial ovarian cancers (EOCs) in the literature. HGSC: high-grade serous carcinoma, CCC: clear-cell carcinoma, ENC: endometroid carcinoma, MUC: mucinous carcinoma.

Article Snippet: Exosomes were further isolated using differential ultracentrifugation (UC) or by commercial exosomal miRNA isolation kits (Norgen Biotek), and miRNAs were analyzed by next generation sequencing (NGS).

Techniques: Countercurrent Chromatography

Journal: Biomedicines

Article Title: Circulating Exosomal miRNAs as Biomarkers in Epithelial Ovarian Cancer

doi: 10.3390/biomedicines9101433

Figure Lengend Snippet: Summary of current clinical trials related to epithelial ovarian cancer that use miRNAs in the circulatory system or tissues as biomarkers. FIGO: Federation of Gynecology and Obstetrics.

Article Snippet: Exosomes were further isolated using differential ultracentrifugation (UC) or by commercial exosomal miRNA isolation kits (Norgen Biotek), and miRNAs were analyzed by next generation sequencing (NGS).

Techniques: Clinical Proteomics, Expressing, Biomarker Discovery, RNA Expression, Blocking Assay

Journal: Avicenna Journal of Medical Biotechnology

Article Title: An Anti-TAZ Monoclonal Antibody Recognizing Cell Surface Expressed TAZ Protein in Human Tumor Cells

doi: 10.18502/ajmb.v15i1.11420

Figure Lengend Snippet: Reactivity of 1F3 with cell surface TAZ on membranes of Burkitt's lymphoma, breast cancer and squamous cell carcinoma lines using flow cytometry technique. Values demonstrate the percentages of positive cells. 1F3 detected cell surface expression of TAZ in pathological conditions with no reactivity in normal conditions. Anti-TAZ mAb was applied on: A) A431, B) MCF-7, C) Raji D) HFFF-PI6 E) MDA-MB-231 G) Normal PBMC (anti-TAZ mAb) and anti-TAZ pAb was used on F) A431 (commercial pAb anti TAZ).

Article Snippet: To evaluate the localization of TAZ protein, flow cytometry analysis was performed both at intracellular and cell surface staining using both produced anti-TAZ mAb and polyclonal anti-TAZ antibody (Abcam).

Techniques: Flow Cytometry, Expressing

Journal: Journal of nanobiotechnology

Article Title: Exosome-derived circUPF2 enhances resistance to targeted therapy by redeploying ferroptosis sensitivity in hepatocellular carcinoma.

doi: 10.1186/s12951-024-02582-6

Figure Lengend Snippet: Fig. 1 Exosomes derived from sorafenib-resistant Huh-7 cells induce an enhanced resistance to sorafenib in HCC cells. (A) Sorafenib-resistant cell line, Huh-7-SR, was established and confirmed by the IC50 value. (B) Co-culture systems were established to investigate the effects of sorafenib-resistant HCC cells on regular HCC cells. The group with additional GW4869 served as a negative control. (C) Cell viabilities of HCC cells co-cultured with Huh-7 or Huh- 7-SR cells under sorafenib stress were evaluated via CCK-8 assays. (D) Assessment of cell proliferation using colony formation assay in Huh-7 and Hep3B cells with the indicated co-culture model. (E) Concentration and size of exosome were measured by NTA. Exosomes were isolated from the supernatant of the culture medium of Huh-7-SR (Exo-SR) and Huh-7 (Exo-Norm) cells by ultracentrifugation. (F) The iconic cup-shaped double-layer closed vesicle structure of isolated exosome was confirmed by TEM. (G) Western blotting analysis of exosomal markers, including CD9, CD63, CD81, LAMP2, TSG101 and Calnexin. An exosome standard from HEK293 cell line was analyzed as a positive control, while Huh-7-SR cell lysate was performed as a negative control. (H) The fluorescent signal of PKH67 labeled Exo-SR was captured in Huh-7 and Hep3B cells. (I) CCK-8 assays were performed to assess the cell viability of Huh-7 and Hep3B cells treated with sorafenib (6 µM) while co-cultured with the indicated exosomes. (J) Colony formation assays were performed to assess the cell proliferation of Huh-7 and Hep3B cells. The data are presented as the mean ± SD of at least three independent experiments. ***P < 0.001

Article Snippet: A positive control was conducted using a standard sample of exosomes derived from HEK293 cells (Novus, USA), while a negative control was performed using Huh-7-SR cell lysate.

Techniques: Derivative Assay, Co-Culture Assay, Negative Control, Cell Culture, CCK-8 Assay, Colony Assay, Concentration Assay, Isolation, Western Blot, Positive Control, Labeling