oeNC- or oeFGF23-infected cells were seeded into 6-well plates (5x105 cells/well) and divided into the following groups: OeNC, cells infected with oeNC; oeFGF23, cells infected with oeFGF23; LY294002, cells infected with oeNC and incubated with 10 µM LY294002 (PI3K inhibitor; MedChemExpress) at 37˚C for 12 h (19 (link)); and oeFGF23 + LY294002, cells infected with oeFGF23 and incubated with 10 µM LY294002 at 37˚C for 12 h. Primary neurons without infection or inhibitor treatment were used as the control group. The primary neurons in all groups were then stimulated with 100 µM H2O2 for 24 h at 37˚C. Finally, the cells were harvested for RT-qPCR, western blotting and apoptosis analysis.
Ly294002
Manufactured by MedChemExpress
Sourced in United States, China
LY294002 is a potent and selective phosphoinositide 3-kinase (PI3K) inhibitor. It inhibits the activity of PI3K, a key enzyme involved in cell signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (18 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (11 mentions)
Pd98059, by MedChemExpress (10 mentions)
Rapamycin, by MedChemExpress (10 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (8 mentions)
Sb203580, by MedChemExpress (7 mentions)
Mk2206, by MedChemExpress (6 mentions)
Sch772984, by MedChemExpress (5 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (5 mentions)
Bay 11 7082, by MedChemExpress (4 mentions)
Anti akt, by Cell Signaling Technology (4 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (4 mentions)
Bcl 2, by Cell Signaling Technology (4 mentions)
Sp600125, by MedChemExpress (4 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (4 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (3 mentions)
P akt, by Cell Signaling Technology (3 mentions)
Anti akt, by Abcam (3 mentions)
Buparlisib, by MedChemExpress (3 mentions)
Cleaved caspase 3, by Cell Signaling Technology (3 mentions)
166 protocols using ly294002
1
Investigating the Role of PI3K in FGF23-Mediated Neuronal Oxidative Stress
2
Investigating miR-186-3p and IGF1 in Cervical Cancer
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The cell culture conditions were 37°C with 5% CO2. The human cervical surface epithelial cell line (HcerEpic), human CC cell lines (HeLa, CaSki, SiHa, and C33A), and embryonic kidney cell line (HEK293T) acquired from ATCC (USA) were cultured in a plate with RPMI-1640 medium (Gibco, USA) containing 10% fetal bovine serum and 1% penicillin/streptomycin (Gibco, USA).
The miR-186-3p inhibitor, miR-186-3p mimic, and paired miRNA negative control (inhibitor-NC and mimic-NC) were designed and synthesized by Shanghai Genechem (Shanghai, China). Before transfection, HeLa and SiHa cells were seeded at a density of 5 × 104 cells/well for 12 h. At approximately 40–60% confluence, the cells were transfected with miR-186-3p mimic, miR-186-3p inhibitor, mimic-NC, or inhibitor-NC using Lipofectamine 3000 (Invitrogen, USA). To knock down the expression of IGF1, specific IGF1 interfering oligonucleotides (si-IGF1) were purchased from Shanghai Jikai Gene Chemistry Co. (Shanghai, China). Then, si-IGF1 was used to treat CC cell lines. Nonspecific control siRNA and reagent controls were used in all the experiments. After transfecting the cells for 48 h, the cell suspension was collected for subsequent experiments. For pathway exploration, LY294002 (Medchem Express, USA) was used as a PI3K inhibitor at a concentration of 50 μM. The cells were pretreated with LY294002 for 2 h.
The miR-186-3p inhibitor, miR-186-3p mimic, and paired miRNA negative control (inhibitor-NC and mimic-NC) were designed and synthesized by Shanghai Genechem (Shanghai, China). Before transfection, HeLa and SiHa cells were seeded at a density of 5 × 104 cells/well for 12 h. At approximately 40–60% confluence, the cells were transfected with miR-186-3p mimic, miR-186-3p inhibitor, mimic-NC, or inhibitor-NC using Lipofectamine 3000 (Invitrogen, USA). To knock down the expression of IGF1, specific IGF1 interfering oligonucleotides (si-IGF1) were purchased from Shanghai Jikai Gene Chemistry Co. (Shanghai, China). Then, si-IGF1 was used to treat CC cell lines. Nonspecific control siRNA and reagent controls were used in all the experiments. After transfecting the cells for 48 h, the cell suspension was collected for subsequent experiments. For pathway exploration, LY294002 (Medchem Express, USA) was used as a PI3K inhibitor at a concentration of 50 μM. The cells were pretreated with LY294002 for 2 h.
3
Transwell Assay for Cell Migration
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We used transwell chambers (Corning, NY, USA) with a pore size of 8 μm to detect cell migration. In total, 24 h before the experiment, the medium was changed to a serum-free medium, and the cells were starved and placed in a 37°C, 5% CO2 incubator. The cells were trypsinized and centrifuged. SB203580 (MedChemExpress), Y27632 (MedChemExpress), and LY294002 (MedChemExpress) were diluted in DMEM containing 0.3% BSA at a concentration of 25 nM/ml for SB203580, 10 nM/ml for Y27632, and 10 nM/ml for LY294002. The concentration of the cell suspension was adjusted to 8.0 × 105 cells/ml added to the upper chamber of the Transwell. The intervention group was pretreated by adding the above signal pathway inhibitors in the upper chamber for 30 min, the control group was added with a basal medium in the lower chamber, and the experimental and intervention groups were added with VEGF (40 ng·ml−1) in the lower chamber. After 24 h of incubation at 37°C, the cells that had migrated to the lower surface were fixed with 4% paraformaldehyde for 15 min and stained with 1% crystal violet for 30 min. Then, the migrating cells were photographed with an inverted microscope (×200 magnification) and counted using ImageJ software.
4
Long-term PI3K Inhibitor Treatment
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5
Inhibiting AKT Activity in Skeletal Muscle
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To disrupt AKT activity in cultured SCs, SCs were initially expanded in growth medium and then induced to differentiate by switching growth medium to differentiation medium containing 10 μM LY294002 (Med Chem Express).
To explore the effects of AKT activity inhibition on skeletal muscle regeneration, we first intramuscularly injected LY294002 beginning five days after injury. Then, LY294002 (5 mg/kg) was intraperitoneally injected every two days for a total of four doses. Muscle samples were harvested one day after the last LY294002 injection. The vector used to deliver LY294002 to mice contained 10% DMSO, 40% PEG300, 5% Tween-80 and 45% saline. A vector containing the same volume of DMSO was also injected into the control mice.
To measure the effects of AKT activity inhibition on dystrophic muscular performance, we intraperitoneally injected LY294002 (5 mg/kg) into mdx mice at the age of two months. LY294002 injection was performed every two days for a total of twelve doses. Muscle samples were collected ten days after the last LY294002 injection. A vector containing the same volume of DMSO was also injected into the control mdx mice.
To explore the effects of AKT activity inhibition on skeletal muscle regeneration, we first intramuscularly injected LY294002 beginning five days after injury. Then, LY294002 (5 mg/kg) was intraperitoneally injected every two days for a total of four doses. Muscle samples were harvested one day after the last LY294002 injection. The vector used to deliver LY294002 to mice contained 10% DMSO, 40% PEG300, 5% Tween-80 and 45% saline. A vector containing the same volume of DMSO was also injected into the control mice.
To measure the effects of AKT activity inhibition on dystrophic muscular performance, we intraperitoneally injected LY294002 (5 mg/kg) into mdx mice at the age of two months. LY294002 injection was performed every two days for a total of twelve doses. Muscle samples were collected ten days after the last LY294002 injection. A vector containing the same volume of DMSO was also injected into the control mdx mice.
6
Modulating MSC Survival under Hypoxia
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A total of nine groups were established with different treatments: (1) control group—negative control with no special treatment; (2) siAPJ group—MSCs transfected with siRNA-APJ; (3) siAPJ NC group—MSCs transfected with siRNA-APJ NC; (4) H/I group—MSCs incubated with serum-deprived medium in a hypoxia incubator chamber (STEMCELL, Canada) containing 1% O2, 94% N2, and 5% CO2 for 24 h [28 (link), 29 (link)]; (5) ELA group—MSCs cultivated in serum-deprived medium with 5 μM ELA in a hypoxia incubator chamber for 24 h; (6) siAPJ + ELA group—MSCs transfected with siRNA-APJ before treatment in the ELA group; (7) siAPJ NC + ELA group—MSCs transfected with siRNA-APJ NC before treatment in the ELA group; (8) LY294002 + ELA group—MSCs pretreated with 50 μM LY294002 (PI3K/AKT pathway inhibitor; Med Chem Express, USA) for 2 h prior to ELA; and (9) U0126 + ELA group—MSCs pretreated with 10 μM U0126 (ERK pathway inhibitor; Med Chem Express, USA) for 2 h prior to ELA. It should be noted that MSCs in groups 6 through 9 were then handled under the same process as the ELA group after the abovementioned treatment.
7
Ex4 Protects Astrocytes from OGD-Induced Injury
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Control cultures were incubated with serum-free 4.5 g/L glucose DMEM at 37 °C in a 5% CO2/95% air atmosphere for 4 h and then refresh normal medium with (Ex4 group) or without Ex4(Medium group). Astrocytes were washed twice with PBS and incubated with glucose-free DMEM [oxygen-glucose deprivation (OGD) medium]. Then, cells were transferred to an anaerobic chamber filled with a gas mixture of 95% N2 and 5% CO2 at 37 °C for 2 h or 4 h as the OGD treatment (OGD+RO group). After OGD treatment, a germ-free glucose solution and FBS were added to the OGD medium to yield 4.5 g/L glucose DMEM supplemented with 10% FBS and cells were cultured for an additional 24 h with or without the corresponding treatment [including 10/50/100/200 nM GLP-1R activator Ex-4 (OGD+RO+Ex4 group) and/or 0.1 μM GLP-1R antagonist exendin-(9-39) (Ex[9–39] (OGD+RO+Ex4+Ex[9–39] group)] (MedChemExpress, USA) and/or 10 μM AG490 (OGD+RO+AG490 group) and 20 μM LY294002 (OGD+RO+Ex4+ LY294002 group) at 37 °C in a humidified atmosphere of 5% CO2/95% air.
8
Glucose-Induced Kidney Cell Response
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Rat renal tubular epithelial NRK-52E cells and human embryonic kidney 293 (HEK293) cells were purchased from American Type Culture Collection (ATCC; Manassas, USA) and cultured in Dulbecco’s modified Eagle medium (DMEM, Sigma-Aldrich, St. Louis, USA) containing 5% calf bovine serum (CBS, Sigma-Aldrich, St. Louis, USA) and 0.05% dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, USA) at 37°C.
NRK-52E cells were divided into four groups: normal glucose (NG) group, high glucose (HG) group, HG+DHM group, HG+LY294002 group and HG+rapamycin group. The cells in NG group were stimulated with 5.5 mmol/L glucose, in HG group with 30 mmol/L glucose, in HG+DHM group with 30 mmol/L glucose and 1 µM DHM, in HG+ LY294002 group with 30 mmol/L glucose and PI3K inhibitor LY294002 (MedChemExpress, New Jersey, USA), and in HG+rapamycin group were stimulated with 30 mmol/L glucose and the autophagy inhibitor rapamycin (Acmec Biochemical, Shanghai, China).
MiR-155-5p inhibitor, miR-155-5p mimic NC inhibitor, or NC mimic were transfected into NRK-52E cells at 37°C for 48 h using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, USA).
NRK-52E cells were divided into four groups: normal glucose (NG) group, high glucose (HG) group, HG+DHM group, HG+LY294002 group and HG+rapamycin group. The cells in NG group were stimulated with 5.5 mmol/L glucose, in HG group with 30 mmol/L glucose, in HG+DHM group with 30 mmol/L glucose and 1 µM DHM, in HG+ LY294002 group with 30 mmol/L glucose and PI3K inhibitor LY294002 (MedChemExpress, New Jersey, USA), and in HG+rapamycin group were stimulated with 30 mmol/L glucose and the autophagy inhibitor rapamycin (Acmec Biochemical, Shanghai, China).
MiR-155-5p inhibitor, miR-155-5p mimic NC inhibitor, or NC mimic were transfected into NRK-52E cells at 37°C for 48 h using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, USA).
9
Epithelial V-like Antigen 1 Expression Analysis
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Epithelial V‐like antigen 1 EVA1 and GAPDH antibodies were acquired from Proteintech (11787‐1‐AP); Flag antibody was purchased from Sigma (F3165); AKT and p‐AKT473 were obtained from Cell Signaling Technology (4691 and 4051); pan‐PI3K inhibitor LY294002 was purchased from MedChemExpress (HY‐10108); DMEM was obtained from Gibco (12100046); FBS was acquired from Hyclone (SH30070); and Matrigel was obtained from Corning (356237).
10
Lipid Extraction and Analysis
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Liquid-chromatography-grade acetonitrile, methanol, dichloromethane, and isopropanol were purchased from Fisher Scientific (Pittsburgh, PA, USA). Internal standard TAG 45:0, sodium valproate, Oil Red O powders, and ammonium acetate were purchased from Sigma-Aldrich (St. Louis, MO, USA). GW9662, rosiglitazone, and LY294002 were purchased from MedChemExpress (Shanghai, China). Ultrapure water was prepared using a Milli-Q water purification system procured from Millipore Corp. (Billerica, MA, USA).
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