GW4869 (GW69A) is a selective and non-competitive inhibitor of neutral sphingomyelinase N-SMase (IC50=1 μM). GW4869 also inhibits exosome synthesis/release and is commonly used in exosome-related studies.

METHODS: Human breast cancer cells MCF-7 were pretreated with GW4869 (10 μM) for 30 min, then incubated with TNF (3 nM) for 6-24 h. Ceramide levels were detected by E. coli diacylglycerol kinase assay.
RESULTS: GW4869 significantly inhibited TNF-induced ceramide accumulation, and GW4869 inhibited N-SMase. [1]
METHODS: Macrophage RAW264.7 was pretreated with GW4869 (10-20 μM) for 2 h, and then incubated with LPS (1 μg/mL) for 24 h. AchE activity was measured.
RESULTS: After pretreatment of macrophages with 10 μM GW4869, LPS-triggered exocytosis was significantly attenuated in macrophages, and the activity of AChE was reduced by 22%. Treatment with 20 μM GW4869 further enhanced this attenuation. [2]

METHODS: To detect in vivo activity, GW4869 (2.5 μg/g) was injected intraperitoneally into C57BL/6 mice, and LPS (25 μg/g) was injected 1 h later.
RESULTS: Pretreatment of mice with GW4869 attenuated LPS-triggered production of exosomes and pro-inflammatory cytokines in the blood, thereby reducing myocardial inflammation. [2]
METHODS: To assay in vivo activity, GW4869 (200 μL of 0.3 mg/mL, 2-2.5 μg/g) was administered intraperitoneally to 5XFAD mice every two days for six weeks.
RESULTS: GW4869 reduced amyloid plaque formation in vivo by blocking exosome secretion. [3]

B. cereu sphosphatidylcholine-PLC is incubated in the presence or absence of 10 μM GW4869 in a reaction mixture containing 100 mM Tris, pH 7.2, 25% glycerol, 20 mM p-nitrophenyl/phosphorylcholine, and production of p-nitrophenol is quantified spectrophotometrically at 410 nm. Protein phosphatase 2A from bovine kidney is incubated in the presence or absence of GW4869 in buffer containing 50 mM Tris, pH 7.4, 1 mM dithiothreitol, 100 μM MnCl2, and 20% glycerol, and phosphatase activity is measured[1].

GW4869 is routinely stored at ?80?°C as a 1.5 mM stock suspension in Me2SO. Right before use, the suspension is solubilized by the addition of 5% methane sulfonic acid (MSA) (2.5 μl of 5% MSA in sterile double-distilled Water are added to 50 μL of GW4869 stock suspension). Cells are treated with GW4869 for 30 min and then TNF is added in 10 μL/well. At the indicated time points, 25 μL of MTT stock solution are added to each well and incubated at 37?°C in 5% CO2 for 3 h. The cell viability is using the MTT assay[1].