Journal: Frontiers in Cell and Developmental Biology

Article Title: Hepatoma Cell-Derived Extracellular Vesicles Promote Liver Cancer Metastasis by Inducing the Differentiation of Bone Marrow Stem Cells Through microRNA-181d-5p and the FAK/Src Pathway

doi: 10.3389/fcell.2021.607001

Figure Lengend Snippet: Identification of BMSCs and hepatoma cell-derived EVs. (A) The morphology of EVs was observed by transmission electron microscopy. (B) The size and concentration of EVs were analyzed by nanoparticle tracking, the size was 110 ± 11 nm; the concentration was 4 × 10 6 particles/mL. (C) Western blot analysis detected the expression of specific marker proteins, and the conditioned medium of HepG2 cells treated with EV inhibitor GW4869 was used as the control. (D) The morphology of primary BMSCs was observed under an optic microscope. (E) The surface marker proteins of BMSCs was detected by flow cytometry, CD29/CD44/CD105 was positive, while CD34/CD45/HLA-DR was negative. (F) The differentiation potency of BMSCs was detected; from left to right, it is osteogenic differentiation, lipogenic differentiation, and chondrogenic differentiation. Stained color implied positive. Replicates = 3.

Article Snippet: Western bolt analysis was utilized to determine the specific markers ( ) of the EVs, and the conditioned medium (CM) supplemented with EV inhibitor GW4869 (MCE, Monmouth Junction, NJ, United States) served as the control. miR-181d-5p mimic, miR-NC, miR-181d-5p inhibitor were synthesized by GeneChem (Shanghai, China).

Techniques: Derivative Assay, Transmission Assay, Electron Microscopy, Concentration Assay, Western Blot, Expressing, Marker, Control, Microscopy, Flow Cytometry, Staining

Journal: Cancer Immunology Research

Article Title: Extracellular Vesicle–Packaged circATP2B4 Mediates M2 Macrophage Polarization via miR-532-3p/SREBF1 Axis to Promote Epithelial Ovarian Cancer Metastasis

doi: 10.1158/2326-6066.CIR-22-0410

Figure Lengend Snippet: EV-packaged circATP2B4 facilitates the peritoneum metastasis of EOCs via inducing M2 macrophage in vivo . A, SKOV3 co-injected with macrophages pelvic peritoneal invasion in nude BALB/C mice. Arrows show tumors in the abdominal cavity, ( n = 5 per group). B and C, Tumor weight and nodules counts analysis of nude mice intraperitoneal metastasis model. D, FISH analysis of circATP2B4, IHC analysis of E-cadherin, N-cadherin, CA125 and LDHA proteins. Scale bars, 50 μm. E, ID8 and EVs inhibitor (GW4869) injected pelvic peritoneal invasion in C57BL/6 mice. Arrows show tumors in the abdominal cavity. F and G, Tumor weight and nodules counts analysis of C57BL/6 mice intraperitoneal metastasis model, ( n = 5 per group). H, Schematic model of EV-packaged circATP2B4 mediating M2 macrophage polarization via circATP2B4/miR-532-3p/SREBF1, which facilitates the EMT, migration, invasion, and metastatic potential of ovarian cancer cells.*, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: GW4869 (inhibitor of extracellular vesicles release, TargetMol) was initially dissolved in DMSO into a stock solution of 100 mmol/L before dilution in PBS to 20 μmol/L for final injection and final DMSO concentration of 0.01%.

Techniques: In Vivo, Injection, Migration

Journal: Autoimmunity

Article Title: CAFs-released exosomal CREB1 promotes cell progression and immune evasion in thyroid cancer via the positive regulation of CCL20.

doi: 10.1080/08916934.2025.2458324

Figure Lengend Snippet: Figure 4. CAFs-secreted exosomes carrying CREB1 enhanced CCL20 expression. (A-B) Exosomes isolated from NFs and CAFs were verified by morphology obser vation under TEM (A) and exosomal markers (Alix, CD63, and Tsg101) using western blot (B). (C) NFs and CAFs isolated exosomes were labeled with PKH67 and co-cultured with TC cells, followed by detection of the uptake of exosomes via fluorescence confocal microscopy. (D) CREB1 mRNA expression was quantified by RT-qPCR after TC cells were cultured in NF and CAF media treated with GW4869 or not. (E) Western blot was performed for protein analysis of CREB1 in TC cells incubated with exosomes from CAFs of sh-NC or sh-CREB1 transfection. (F) CCL20 was determined via western blot after TC cells were incubated with exosomes from CAFs of sh-NC, sh-CREB1, sh-CREB1 + pcDNA, sh-CREB1 + CCL20 group. *p < 0.05.

Article Snippet: In addition, TC cells were cultured in NFs and CAFs medium treated with GW4869 (20 μM; Selleck, Houston, TX, USA), and CREB1 mRNA expression was examined by western blot.

Techniques: Expressing, Isolation, Western Blot, Labeling, Cell Culture, Fluorescence, Confocal Microscopy, Quantitative RT-PCR, Incubation, Transfection

Journal: Development (Cambridge, England)

Article Title: SMPD4-mediated sphingolipid metabolism regulates brain and primary cilia development.

doi: 10.1242/dev.202645

Figure Lengend Snippet: Fig. 6. Cilia shortening in SMPD4 models is rescued by in vitro ceramide treatment. (A) Schematic of ceramide synthesis and where compounds used in this study will act. (B-P) iPSCs were treated with PBS, C16 ceramide (CER), GW4869 or FB1. Treatment with GW4869 or FB1 decreases number of cilia in control iPSCs (N; n=8 each control, GW4869 or FB1; ANOVA, P<0.001). Treatment of SMPD4 KO and patient iPSCs with ceramide does not impact the number (O; n=10 control, 8 ceramide treated, P=0.927; 12 knockout, 6 ceramide treated, P=0.668; 12 patient, 6 ceramide treated, P=0.425), but does increase the length of primary cilia per cell (P; n=21 control, 22 ceramide treated, P=0.544; 19 knockout, 30 ceramide treated, P<0.001; and 29 patient, 22 ceramide treated, P<0.001). Vertical bar (N-P) shows median. Scale bars: 20 µm (B-D, for B-M).

Article Snippet: Cells were treated with Fumonisin B1 (Sigma-Aldrich) at 30 μM for 72 h, GW4869 (Tocris Bioscience) at 10 μM for 72 h, C16 ceramide (Avanti Polar Lipids) at 2 μM for 48 h, ACY1215 (APExBIO) at 1 μM for 18 h and/or IWP2 (Tocris Bioscience) at 2 μM for 48 h before being processed for immunocytochemistry.

Techniques: In Vitro, Control, Knock-Out

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