PI3K inhibition by PI828 LY294002 is determined in a radiometric assay using purified, recombinant enzymes (class IA class IB) with 1 μM ATP. The kinase reaction is carried out for 1 h at room temperature (24°C) is terminated by addition of PBS. IC₅₀ values are subsequently determined using a sigmoidal dose-response curve fit (variable slope). CK2 GSK3β (glycogen synthase kinase 3β) inhibition is established by kinase selectivity screening. Inhibitor (10 μM; PI828 LY294002) is tested against the Upstate panel of kinases in 10 μM ATP^[2].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

LY294002 is dissolved in DMSO stored, then diluted with appropriate media (DMSO 0.5%) before use^[3].

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Human nasopharyngeal carcinoma cell line CNE-2Z is seeded 96-well plates at 5000 cells/well. Twenty-four hours after cells are seeded, the medium is removed replaced in the presence of LY294002 (0 μM, 10 μM, 25 μM, 50 μM, 75 μM) dissolved in DMSO DMSO only for an additional 24 h 48 h. To avoid any nonspecific toxic effects of DMSO on cell growth, DMSO concentrations are maintained at 0.5% in all experiments. MTT dye (5 mg/mL) is added to each well. The reaction is stopped by the addition of DMSO, optical density is measured at 490 nm on a multiwell plate reader. Background absorbance of the medium in the absence of cells is subtracted. All samples are assayed in triplicate, the mean for each experiment is calculated. Results are expressed as a percentage of control, which is considered to be 100%^[3].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

LY294002 is dissolved in vehicle (DMSO).

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Mice^[3]
Athymic nude mice are used when they are 6-8 weeks. Mice are randomly divided free separated five groups (n=4 mice). Mice are housed in the same environment with controlled temperature, humidity, a 12 h light/dark cycle. Mice are inoculated subcutaneously with CNE-2Z cells (1×10⁶ cells/mouse in 200 μL of RPMI-1640) the flank. The tumor take rate is 100%. After 1 week, an intraperitoneal injection is performed to the xenograft mice with different dosage of LY294002 (10 mg/kg, 25 mg/kg, 50 mg/kg, 75 mg/kg twice weekly (n=4 mice), each group for 4 weeks. Treated mice are monitored any signs. Body weight tumors size are measured twice a week. Tumor size is measured using calipers tumor volume is calculated (volume=long axis×short axis²). At the end of the treatment, all mice are euthanized. One part of tumor tissue is fixed in formalin embedded in paraffin, another part is stored at -70°C.
Rats^[4]
Male Sprague-Dawley rats weighing 220-240 g are anesthetized by intraperitoneally injecting pentobarbital sodium (50 mg/kg). The animals are divided 3 groups: NMDA+vehicle (DMSO) (n=46), NMDA+LY294002 (50 nmol) (n=25), NMDA+Wortmannin (50 nmol) (n=23). Either LY294002 wortmannin mixed with 200 nmol of NMDA in a total volume of 5 μL is injected the vitreous cavity of one eye. The same volume of DMSO is injected the vitreous cavity of the contralateral eye, which is used as a control. The injections are performed under a microscope using a 32-gauge needle, which is connected to a microsyringe. The needle is inserted approximately 1 mm behind the corneal limbus. Damage to neurons blood vessels in the retina is assessed at 2 7 days after the injection. The effects of the intravitreal treatment with either LY294002 Wortmannin alone on retinal neurons blood vessels are also examined.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Chaussade C, et al. Evidence for functional redundancy of class IA PI3K isoforms in insulin signalling. Biochem J. 2007 Jun 15;404(3):449-58.

  [2]. Gharbi SI, et al. Exploring the specificity of the PI3K family inhibitor LY294002. Biochem J. 2007 May 15;404(1):15-21.

  [3]. Jiang H, et al. Phosphatidylinositol 3-kinase inhibitor(LY294002) induces apoptosis of human nasopharyngeal carcinoma invitro in vivo. J Exp Clin Cancer Res. 2010 Apr 22;29:34.

  [4]. Ueda K, et al. Differential effects of LY294002 wortmannin on neurons vascular endothelial cells in the rat retina. Pharmacol Rep. 2013;65(4):854-62.

  [5]. Wang X, et al. Remote Ischemic Postconditioning Protects against Myocardial Ischemia-Reperfusion Injury by Inhibition of the RAGE-HMGB1 Pathway. Biomed Res Int. 2018 Jan 23;2018:4565630.

307.34

C₁₉H₁₇NO₃

154447-36-6

O=C1C=C(OC2=C1C=CC=C2C3=CC=CC=C3)N4CCOCC4

Powder	-20°C	3 years
	4°C	2 years
In solvent	-80°C	6 months
	-20°C	1 month

Room temperature in continental US; may vary elsewhere