Journal: Cell Communication and Signaling : CCS

Article Title: Cancer-associated adipocytes promote peritoneal metastasis of colorectal signet ring cell carcinoma via FABP4 induction

doi: 10.1186/s12964-025-02569-2

Figure Lengend Snippet: CAA-derived exosomes mediate FABP4 induction and promote SRCC stemness and metastasis. A Transmission electron microscopy (TEM) image of exosomes isolated from CAAs and NAs. B Western blot analysis of exosomal protein markers CD63, CD81, and TSG101 in EVs isolated from NAs and CAAs. Marker expression confirmed the identity of the isolated EVs. C Western blot showing FABP4 upregulation in PDOs treated with CAA-derived exosomes. Western blot analyses were performed in three independent biological replicates, and representative results are presented. D FABP4 induction was blocked by exosome release inhibitor GW4869. Western blot analyses were performed in three independent biological replicates, and representative results are presented. E–H GW4869 treatment reversed CAA-induced upregulation of stemness and EMT markers. I–K GW4869 significantly suppressed CAA-induced PDO spheroid formation, proliferation, and peritoneal metastasis. L Schematic depiction of the promotion on peritoneal metastasis by exosomal FABP4 derived from CAAs in SRCC. All quantifications are shown as mean ± SD from three independent biological replicates ( n = 3). * p < 0.05 by unpaired t-test

Article Snippet: For inhibition experiments, exosome secretion was blocked using GW4869 (Selleckchem, #S7609) at a final concentration of 10 μM for 24 h.

Techniques: Derivative Assay, Transmission Assay, Electron Microscopy, Isolation, Western Blot, Marker, Expressing

Journal: Oncotarget

Article Title: Elimination of LMP1-expressing cells from a monolayer of gastric cancer AGS cells

doi: 10.18632/oncotarget.16996

Figure Lengend Snippet: (A) IL-8 expression was upregulated in AGS-RFP/LMP1 and AGS cell co-cultures. AGS-RFP/LMP1 or AGS-RFP cells were mixed with AGS cells at a ratio of 1:24 and cultured for 48 h in the presence of GW4869 (10 μM) or DMSO. RNA was extracted and subjected to qRT-PCR. Values are expressed as the fold change relative to that of the control after normalization to the housekeeping gene GAPDH. Data are means ± standard error from three to six independent experiments. ** P < 0.01; * P < 0.05. (B) ADAM10 expression was upregulated in AGS-RFP/LMP1 and AGS cell co-cultures. Samples were prepared as described in Figure . Data are presented as means ± standard error from three independent experiments. * P < 0.05. (C) The effect of IL-8 on EGFR phosphorylation in AGS cells was induced by co-culturing with AGS-RFP/LMP1 cells. Cells were preincubated for 30 min with 8 μg/ml anti-IL-8 (to neutralize IL-8) or control antibody and then treated with CM to induce EGFR phosphorylation. The band intensities of phosphorylated EGFR were quantified and normalized to those of EGFR. * P < 0.05. ( D and E ) LMP1-positive cells produced exosomes containing LMP1. Exosomes were isolated from the supernatant of LMP1-positive or -negative cells (D) and from the supernatant of AGS-RFP/LMP1 cells in the presence of GW4869 (10 μM) or DMSO (E) . Hsp70 was used as an exosome marker. (F) LMP1-containing exosomes secreted from LMP1-positive cells spread to surrounding LMP1-negative cells. AGS-EGFP/LMP1 cells were mixed with AGS cells at a ratio of 2:98 and then cultured on coverslips in the presence of GW4869 (10 μM) or DMSO. Cells were fixed and stained for LMP1 and EGFP using specific antibodies. Nuclei were stained with DAPI. Arrowheads indicate transported LMP1 in surrounding AGS cells.

Article Snippet: For inhibition of exosome secretion, cells were treated with 10 μM GW4869 (Cayman) for 24–48 h before harvesting. (S)-(−)-blebbistatin (30 μM; Toronto Research Chemicals), Y27632 (10 μM; Cayman) and ML-7 (10 μM; Cayman) were used to inhibit the Rho/myosin-II pathway.

Techniques: Expressing, Cell Culture, Quantitative RT-PCR, Produced, Isolation, Marker, Staining

Journal: European Journal of Medical Research

Article Title: BMSC-derived exosomes protect against kidney injury through regulating klotho in 5/6 nephrectomy rats

doi: 10.1186/s40001-022-00742-8

Figure Lengend Snippet: Effect of exosome transplantation on klotho activation and expression. A Fluorescent report plasmid containing klotho promoter and pRL-TK were co-transfected into 293 T cells. The transfected cells were randomly divided into five groups: control group (PBS), TGF-β1 group (1 ng/μL TGF-β1), BMSCs group (1 ng/μL TGF-β1+co-cultured with 5 × 10 4 well BMSCs), exosomes (1 ng/μL TGF-β1+0.4 mg exosomes), and exosome inhibitor group (1 ng/μL TGF-β1+co-cultured with 5 × 10 4 well BMSCs+GW4869). Then the firefly and Renilla luciferase activities were measured using the dual luciferase reporter gene assay. Data are shown as mean ± SD ( n = 3). * p < 0.05, ** p < 0.01. The protein expression of klotho was also evaluated using western blotting B , C qRT-PCR were performed to detect the mRNA expression of klotho. mRNA expression levels were normalized to that of GAPDH. Quantitative data ( n = 6–7) are provided as the mean ± SD. * p < 0.05, ** p < 0.01. D Expression of klotho protein in the control, 5/6 Nx, and exosome-treated 5/6 Nx rats was evaluated using western blotting

Article Snippet: The transfected cells were treated with TGF-β1 (1 ng/μL), exosomes (0.4 mg), or co-cultured with BMSCs (5 × 10 4 /well) in a Transwell plate for 24 h. GW4869 (GLPBIO, Montclair, CA, USA) was used as an inhibitor of exosome biogenesis and release.

Techniques: Transplantation Assay, Activation Assay, Expressing, Plasmid Preparation, Transfection, Control, Cell Culture, Luciferase, Reporter Gene Assay, Western Blot, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: Extracellular vesicles package dsDNA to aggravate Crohn’s disease by activating the STING pathway

doi: 10.1038/s41419-021-04101-z

Figure Lengend Snippet: A – C Classification of EVs in colon lavage of murine colitis. A Western blot of specific EVs markers: The simultaneous expression of three classic positive markers of EVs including Alix, CD63, and CD81, along with the absence of Calnexin, together identified EVs. The whole-cell lysate of CT26, a murine cell line, was used as a positive control. B Nanoparticle-tracking analysis of diluted EVs fractions was determined by ZetaView PMX 110 (Particle Metrix, Meerbusch, Germany). The distribution of EVs particle concentration ( y axis) by size ( x axis) was shown and 126.2-nm particles accounted for the highest proportion (98.5%). C Representative transmission electron microscope images of EVs fractions. The yellow arrow pointed to examples of EVs, which were cup-shape membrane-enclosed particles with diameters of 30–150 nm. D Absolute quantification of nDNA and mtDNA within EVs extracted from plasma and colon lavage of murine colitis. Hist1h3F gene and mtCOI gene were applied to represent nDNA and mtDNA respectively. Exosomal mtDNA and nDNA were significantly higher in the plasma and colon lavage of murine colitis. Murine colitis treated with GW4869 were also examined. n = 5–10/group. In the illustrations, healthy wild-type model, murine colitis, and murine colitis treated with GW4869 were abbreviated as WT, WT + DSS, and WT + DSS (GW4869 IP) respectively. Colon lavage was abbreviated as CL. E Classification of EVs isolated from plasma of active human CD, including western blot, nanoparticle-tracking analysis, and representative transmission electron microscope images. In the nanoparticle-tracking analysis, 117.7-nm particles accounted for the highest proportion (98.6%). In the representative transmission electron microscope image, the yellow arrow pointed to EVs. F Levels of exosomal nDNA and mtDNA from the plasma of CD patients were measured ( n = 5/group). Both were significantly higher in patients with active CD. H3 clustered histone 7 gene and mtCOI gene were applied to represent nDNA and mtDNA respectively. G Correlations between the disease activity and levels of exosomal dsDNA in plasma in murine colitis and CD patients (Spearman’s rank correlation analysis). Disease activity index, Crohn’s Disease Activity Index, exosomal nDNA, and exosomal mtDNA were abbreviated as DAI, CDAI, exo-nDNA, and exo-mtDNA in the illustrations respectively. Data were displayed as mean values ± SD at least three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: To inhibit the release of EVs during the modeling period, mice were injected 2.5 mg/kg GW4869 (M4974, AbMole BioScience) intraperitoneally on days 1, 3, and 5.

Techniques: Western Blot, Expressing, Positive Control, Concentration Assay, Transmission Assay, Microscopy, Membrane, Quantitative Proteomics, Clinical Proteomics, Isolation, Activity Assay

Journal: Cell Death & Disease

Article Title: Extracellular vesicles package dsDNA to aggravate Crohn’s disease by activating the STING pathway

doi: 10.1038/s41419-021-04101-z

Figure Lengend Snippet: A – D The expressions of p-STING, STING, p-IRF3, IRF3, p-p65, and p65 in colon tissues from Wild type (WT), STING −/− , WT murine colitis, WT murine colitis treated with GW4869, and STING −/− murine colitis models were determined by western blot. WT murine colitis was abbreviated as WT + DSS in the illustrations. WT murine colitis treated with GW4869 was abbreviated as WT + DSS (GW4869 IP). STING −/− murine colitis was abbreviated as WT + DSS. The activation of the STING pathway was observed in the WT murine colitis model and was inhibited in WT murine colitis treated with GW4869. E , F The proportion of TUNEL-positive cells was quantified to estimate intestinal apoptotic level in WT, WT murine colitis, WT murine colitis treated with GW4869, and STING −/− murine colitis models. The level of apoptosis was higher in the WT murine colitis model and lower in WT murine colitis treated with GW4869, and STING −/− murine colitis models. Representative images were shown. Scale bar = 50 µm. G The expression of inflammatory cytokines including IL-6, TNF-α, and IFN-β in the homogenates of murine colon tissues was measured by ELISA ( n = 5–9/group). All results were representative of at least three independent experiments. Data were displayed as mean values ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: To inhibit the release of EVs during the modeling period, mice were injected 2.5 mg/kg GW4869 (M4974, AbMole BioScience) intraperitoneally on days 1, 3, and 5.

Techniques: Western Blot, Activation Assay, TUNEL Assay, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Cell Death & Disease

Article Title: Extracellular vesicles package dsDNA to aggravate Crohn’s disease by activating the STING pathway

doi: 10.1038/s41419-021-04101-z

Figure Lengend Snippet: A – E Comparison of disease prognosis among six experimental murine models ( n = 11–20/group). WT (GW4869 IP): to examine possible side effects of GW4869, GW4869 was administrated in normal wild-type mice. WT + DSS (GW4869 IP): Murine colitis models treated with GW4869. STING − /− + DSS: STING knockout (STING −/− ) mice were employed and induced by DSS to establish colitis. Indicators of disease activity and severity were examined including A Disease activity index (DAI), B percent changes in body weight, C survival rate, D lengths and representative images of colons, E histological score of colons and representative colon H&E images. Data were displayed as mean values ± SD at least three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: To inhibit the release of EVs during the modeling period, mice were injected 2.5 mg/kg GW4869 (M4974, AbMole BioScience) intraperitoneally on days 1, 3, and 5.

Techniques: Comparison, Knock-Out, Activity Assay