Fresh RAW264.7 macrophages were plated in 100 mm petri dishes at 1.2×106 cells/dish, and treated with either culture media containing 20 μg of exosomes isolated from non-LPS treated macrophages (non-LPS exosomes) or 20 μg of exosomes isolated from LPS treated macrophages (LPS exosomes), or exosome-free media. The culture supernatants were harvested for cytokine ELISA assays at 10 min and 24 h. For inhibition of exosome generation, macrophages were pre-treated with either culture media containing 10 μM or 20 μM GW4869 (Sigma) for 2 h prior to treatment with 1 μg/ml LPS incubation, Culture supernatants were collected after 24 h for AChE activity assay and cytokine measurement. GW4869 was initially dissolved in DMSO (Fisher Scientific) into a stock solution of 5 mM GW4869 before dilution in culture supernatant to achieve 10 μM or 20 μM GW4869 concentration in cell culture condition (Note: final DMSO concentration is 0.005%). To determine the possible toxicity of DMSO or GW4869, fresh RAW 264.7 macrophages (1.2×106 cells/dish) were incubated in culture media containing 0.005% DMSO, 10 μM GW4869 and 20 μM GW4869 for 24 h. Cell injury was determined by measuring the release of lactase dehydrogenase (LDH) into the culture media, using a LDH detection kit (Sigma) according to the manufacturer’s protocol.
Gw4869
Manufactured by Merck Group
Sourced in United States, Germany, China, Macao, United Kingdom
GW4869 is a laboratory reagent manufactured by Merck Group. It is a small molecule inhibitor that functions as a neutral sphingomyelinase 2 (nSMase2) inhibitor. The core function of GW4869 is to inhibit the activity of nSMase2, an enzyme involved in the metabolism of sphingolipids.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (14 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (9 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (7 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (7 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (6 mentions)
Mg132, by Merck Group (4 mentions)
Spiroepoxide, by Santa Cruz Biotechnology (4 mentions)
Bafilomycin a1, by Merck Group (4 mentions)
Exosome free fbs, by System Biosciences (4 mentions)
Cycloheximide (chx), by Merck Group (4 mentions)
Opti mem, by Thermo Fisher Scientific (4 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Pkh67, by Merck Group (3 mentions)
Lipopolysaccharides (lps), by Merck Group (3 mentions)
Egm 2, by Lonza (3 mentions)
Chloroquine, by Merck Group (3 mentions)
Penicillin, by Thermo Fisher Scientific (3 mentions)
Streptomycin, by Thermo Fisher Scientific (3 mentions)
Tsg101, by Abcam (3 mentions)
Calnexin, by Abcam (3 mentions)
252 protocols using gw4869
1
Exosome-mediated Macrophage Activation and Inhibition
2
Isolation and Characterization of MSC Exosomes
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MSCs were maintained in MSC media as described above. After growing to 80% confluence, MSC cells were washed with PBS and then cultured in serum-free DMEM for 24 h, followed by the isolation of MSC exosomes from the supernatant of the cultures with ExoQuick exosome precipitation solution (System Bioscience, Mountain View, CA). The exosomes were quantified using the BCA assay, size was measured by nanoparticle tracking analysis, and the morphological characteristics were determined by transmission electron microscopy (TEM) (Figure S1B–E ).
To demonstrate that MSC-derived exosomes were taken up by cultured HK-2 cells, the latter were incubated with PKH26-labeled exosomes for 24 h and observed under a confocal fluorescent microscope. The method of labeling exosomes was performed according to a previous study [13 (link)].
For exosomal inhibition, MSC cells were cultured in an MSC medium supplemented with 20 μM GW4869 (Sigma) (MSC supernatant+GW4869) for 24 h and then replaced with serum-free MSC medium containing 20 μM GW4869 for 24 h prior to exosome isolation.
To demonstrate that MSC-derived exosomes were taken up by cultured HK-2 cells, the latter were incubated with PKH26-labeled exosomes for 24 h and observed under a confocal fluorescent microscope. The method of labeling exosomes was performed according to a previous study [13 (link)].
For exosomal inhibition, MSC cells were cultured in an MSC medium supplemented with 20 μM GW4869 (Sigma) (MSC supernatant+GW4869) for 24 h and then replaced with serum-free MSC medium containing 20 μM GW4869 for 24 h prior to exosome isolation.
3
Inhibiting EV Secretion Impacts NSC Differentiation
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To investigate the effect of inhibiting the secretion of UTX KO SCMECs-derived EVs (KO EVs) or NC SCMECs-derived EVs (NC EVs) on NSCs differentiation, we need to co-culture SCMECs and NSCs. This study used a 24-well plate with 0.4 μm transwell chamber (Corning, 3413) to establish a co-culture system for SCMECs and NSCs. The SCMECs were seeded in the upper chamber of the transwell. In order to inhibit the secretion of SCMECs EVs, the upper complete medium of the experimental group contained 0.01% GW4869 (Sigma, D1692, GW4869 group), while the control group did not contain GW4869. GW4869 is an inhibitor of sphingomyelinase which can inhibit the biogenesis and release of EVs [37 (link)]. NSCs were digested into single cells and seeded in the lower chamber to construct the SCMECs-NSCs co-culture system.
4
Exosome Secretion Inhibition in CAFs
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To further verify the role of exosomes, exosome secretion was inhibited using Sphingomyelinase inhibitor GW4869 and 10 µM of GW4869 (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) was used to treat CAFs (designated GW4869-CAFs group), while Control group was treated with DMSO (designated DMSO-CAFs group). After 12 h, cells were washed thrice with sterile PBS before 10 ml of serum-free DMEM (Gibco, Grand Island, NY, USA) was added. CAFs were collected after 2 h.
5
Investigating Exosome Inhibition in Lung Exposure
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Mice were ordered from Taconic Biosciences (Albany, NY, USA) and acclimated in appropriate housing facilities for 2 weeks, as per the university-approved institutional animal care and use committee (IACUC) protocol. C57BL/6 mice were randomly assigned to three different treatment groups (n = 10 mice per group): HEPA-filtered air (Lab Products LLC, Seaford, DE) + saline (FA), particulate matter + saline (PM), or PM + an exosome inhibitor, GW4869 (MilliporeSigma, St. Louis, MO, USA) (PM + GW4869) (Figure 1 A). Firstly, 50 μL of 10 μM GW4869 or 1× physiological saline solution (PSS, MilliporeSigma, St. Louis, MO) was instilled into the lungs of mice 1 h prior to inhaled exposure of mine-dust from St. Anthony mine PM exposure or FA prior to each 4 h exposure.
6
Modulating Aβ-induced Endothelial Dysfunction
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Human cerebral microvascular endothelial (hCMEC/D3) cells (Cat# SCC066, RRID: CVCL_U985; Weksler et al., 2005) were purchased from MilliporeSigma and maintained in Endothelial Cell Medium containing 5% fetal bovine serum and 1% penicillin-streptomycin (ScienCell, San Diego, CA, USA, Cat# 1001).
hCMEC/D3 cells were exposed to the following conditions: Aβ1–42 (MilliporeSigma, Cat# A9810), incubated with 1–10 µM Aβ1–42 for 24 hours; Aβ1–42 + GW4869 (nSMase inhibitor, Selleck, Houston, TX, USA, Cat# S7609; Menck et al., 2017), incubated with 10 µM Aβ1–42 and 10 µM GW4869 for 24 hours; verapamil (P-gp inhibitor, MilliporeSigma, Cat# V4629), incubated with 10 µM verapamil for 24 hours; OE-nc, transfected with pcDNA3.1(+) empty vector (RRID: Addgene_47388) for 24 hours; OE-nSMase1, transfected with pcDNA3.1(+)-nSMase1 vector for 24 hours; si-nc, transfected with negative control siRNA for 48 hours; si-nSMase1, transfected with nSMase1-specific siRNA for 48 hours; OE-nSMase1 + GW4869, transfected with pcDNA3.1(+)-nSMase1 vector for 24 hours, followed by treatment with 10 µM GW4869 for 24 hours; and si-nSMase1 + ceramide (MilliporeSigma, Cat# 22244), transfected with nSMase1-specific siRNA for 48 hours, followed by treatment with 10 µM ceramide for 24 hours. The siRNA sequences are shown inAdditional Table 1
hCMEC/D3 cells were exposed to the following conditions: Aβ1–42 (MilliporeSigma, Cat# A9810), incubated with 1–10 µM Aβ1–42 for 24 hours; Aβ1–42 + GW4869 (nSMase inhibitor, Selleck, Houston, TX, USA, Cat# S7609; Menck et al., 2017), incubated with 10 µM Aβ1–42 and 10 µM GW4869 for 24 hours; verapamil (P-gp inhibitor, MilliporeSigma, Cat# V4629), incubated with 10 µM verapamil for 24 hours; OE-nc, transfected with pcDNA3.1(+) empty vector (RRID: Addgene_47388) for 24 hours; OE-nSMase1, transfected with pcDNA3.1(+)-nSMase1 vector for 24 hours; si-nc, transfected with negative control siRNA for 48 hours; si-nSMase1, transfected with nSMase1-specific siRNA for 48 hours; OE-nSMase1 + GW4869, transfected with pcDNA3.1(+)-nSMase1 vector for 24 hours, followed by treatment with 10 µM GW4869 for 24 hours; and si-nSMase1 + ceramide (MilliporeSigma, Cat# 22244), transfected with nSMase1-specific siRNA for 48 hours, followed by treatment with 10 µM ceramide for 24 hours. The siRNA sequences are shown in
7
In vivo Treatment of Mice with Exosome Inhibitor
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For in vivo treatment of mice with the exosome maturation inhibitor GW4869 (Sigma), 8-12 weeks old C57BL/6N mice were injected intraperitoneally with GW4869 (0.3125 or 1.25µg per gram bodyweight) or carrier control once per day for five consecutive days. On day six, mice were intraperitoneally infected with 1x106L. monocytogenes (strain LO28). Bacterial inoculum was prepared as previously described 45 (link). At 24 hours p.i., mice were sacrificed and spleens were harvested. For cfu assays, livers and spleens were weighed, homogenized in PBS, and 1:10 serial dilutions were plated on Oxford agar plates (Merck). Colonies were counted after approximately 30 hours at 37°C. Some mice were treated with ODN1826 (InvivoGen) at a dose of 10 μg per mouse. Animals were housed under specific pathogen-free conditions according to FELASA guidelines and animal experiments have been approved by the Vienna University of Veterinary Medicine institutional ethics committee and performed according to protocols approved by the Austrian law (BMWF 68.205/0032-WF/II/3b/2014).
8
In vivo Treatment of Mice with Exosome Inhibitor
9
Isolation and Characterization of Extracellular Vesicles
10
Inhibition of EV Secretion in THP-1 Cells
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