PC12 cells were adjusted to 1 × 103 to 5 × 103 cells/well on a 96-well plate and cultured for 24 hours, and 5 μM CHIR99021 (252917-06-9, FUJIFILM Wako Pure Chemical Corporation), 5 μM LY294002 (154447-36-6, FUJIFILM Wako Pure Chemical Corporation), 2.5 μM H89, 2.5 μM U0126, 2.5 μM GF109203X, 10 μM Y27632, and 5 μM purvalanol A were added and placed in a CO2 incubator (37°C, 5%) for 1 hour, and PACAP 10−7 M was added postincubation. After culturing for 3 days, the culture was observed using an optical microscope BZ-X710 (Keyence) and the total number of cells and the number of cells producing protrusions (greater than 20 μm and 20 μm or less) were measured.
Ly294002
Manufactured by Fujifilm
Sourced in Japan, United States
LY294002 is a laboratory equipment product manufactured by Fujifilm. It is a chemical compound that functions as a specific inhibitor of phosphoinositide 3-kinase (PI3K), an enzyme involved in various cellular processes. The core function of LY294002 is to serve as a research tool for studying the role of PI3K in biological systems.
Automatically generated - may contain errors
Lab products found in correlation
U0126, by Fujifilm (5 mentions)
Sp600125, by Fujifilm (3 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
U73122, by Cayman Chemical (2 mentions)
Syringe filters, by Iwaki (2 mentions)
Rapamycin, by Fujifilm (2 mentions)
Bz x710, by Keyence (1 mentions)
Chir99021, by Fujifilm (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Pyruvate, by Merck Group (1 mentions)
2 mercaptoethanol, by Fujifilm (1 mentions)
Fetal calf serum (fcs), by Fujifilm (1 mentions)
Glutamine, by Fujifilm (1 mentions)
Non essential amino acid (neaa), by Fujifilm (1 mentions)
Dmem hamf12 medium, by Fujifilm (1 mentions)
Streptozotocin (stz), by Merck Group (1 mentions)
Aldosterone, by Merck Group (1 mentions)
Triamcinolone acetonide, by Fujifilm (1 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (1 mentions)
Ru486, by Cayman Chemical (1 mentions)
40 protocols using ly294002
1
Neurite Outgrowth in PC12 Cells
2
Stem Cell Culture and Inducible Transgene Expression
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The mouse ES cell line E14-Tg2a and that expressing the syntaxin-4 transgene (ES-STstx4) were cultured in GMEM (Wako) containing 10% FCS (Invitrogen), 1 mM glutamine (Wako), non-essential amino acid (Wako), 0.1 mM 2-mercaptoethanol (Wako), 1 mM pyruvate (Sigma) and LIF (Wako). To maintain stemness, inhibitors of GSK3β/MEK1/2 (2i) (CHIR99021/PD0325901; Invitrogen) dissolved in DMSO were added to the medium at a concentration of 2 μM. The mouse EC cell line F9 (ATCC CRL-1720) and its derivatives (F9-STstx4 and F9-Pcadherin) were maintained in DMEM/HamF12 medium (Wako) supplemented with 10% FCS. The P19CL6 cell line (RIKEN BRL RCB2318) and its derivative (P19-STstx4) were cultured in Alpha modified MEM (αMEM; Wako) with 5% FCS. To induce transgene expression, ES-STstx4, F9-STstx4, F9-Pcadherin, or P19-STstx4 was treated with 5 μg/ml Dox (Sigma-Aldrich) for two or three days. In some cultures, an inhibitor of PI3K (LY294002; Wako) was dissolved in DMSO and added at a concentration of 1.25 or 2.5 μM.
3
Müller Glial Cell Line Characterization
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The human Müller glial cell line Moorfields/Institute of Ophthalmology‐Müller 1 (MIO‐M1) was provided from Dr G. Astrid Limb (UCL Institute of Ophthalmology).10 The cells were cultured in DMEM containing 10% FBS (Thermo Fisher Scientific). Twenty‐four hours after transfection, the composite transfection mixture was removed and replaced with 1% FBS/DMEM for 24 hours, followed with recombinant protein and reagent treatments before each assay. Recombinant human IL‐1β proteins were purchased from R&D systems. Aldosterone and streptozotocin (STZ) were from Sigma‐Aldrich. RU486 was from Cayman Chemical. Dexamethasone sodium phosphate, triamcinolone acetonide and LY294002 (PI3K inhibitor) were from FUJIFILM Wako Pure Chemical Corporation. U0126 (ERK1/2 inhibitor) was from Promega. Reagents were dissolved in either PBS, ethanol (final concentration < 0.1%) or dimethyl sulfoxide (DMSO, final concentration < 0.1%).
Specific siRNAs against dual specificity phosphatase (DUSP)1 (hs.Ri.DUSP1.13.3), TSC22 domain family member (TSC22D)3 siRNA (hs.Ri.TSC22D3.13.1) and a negative control siRNA oligo (DS NC1) were purchased from Integrated DNA Technologies and used at 10 nmol/L. Cells were transfected with siRNA using Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific) following the manufacturer's protocols.
Specific siRNAs against dual specificity phosphatase (DUSP)1 (hs.Ri.DUSP1.13.3), TSC22 domain family member (TSC22D)3 siRNA (hs.Ri.TSC22D3.13.1) and a negative control siRNA oligo (DS NC1) were purchased from Integrated DNA Technologies and used at 10 nmol/L. Cells were transfected with siRNA using Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific) following the manufacturer's protocols.
4
Regulation of Primary Mouse Hepatocytes
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Primary mouse hepatocytes were cultured in William’s medium E (Thermo Fisher Scientific) supplemented with 10% foetal bovine serum at 37 °C in 5% CO2. Cells were treated with either an adenovirus (ViraPower Adenoviral Gateway Expression Kit, Thermo Fisher Scientific) expressing mouse Nrg1α or siRNA (Supplemental Table 1 , Integrated DNA Technologies KK, Tokyo, Japan) targeting for mouse Nrg1 24 h before stimulation with 500 μM 8-bromo-cAMP (Cayman Chemical, Ann Arbor, MI, USA) and 100 nM dexamethasone (Sigma) for 3 h. Viruses encoding β-galactosidase were used as a control. A multiplicity of infection of 50 for each virus was used for the infection. In some experiments, cells were pretreated with rNRG1α (1 or 10 nM) 30 min, or inhibitors such as Ly294002 (Wako Pure Chemical Industries), PD98059 (Cayman Chemical) or lapatinib (BioVision, Inc, Milpitas, CA, USA) 1 h before the stimulation. The conditioned media (24 h incubation) of isolated hepatocytes were collected to determine whether the N-terminal region of NRG1 endogenously expressed in hepatocytes was cleaved and released.
5
Pharmacological Targeting of the PI3K/Akt Pathway
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Bevacizumab was purchased from Chugai Pharmaceutical Co. (Tokyo, Japan). LY294002 and VEGFA were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). AS1842856 was purchased from Calbiochem (Merck Millipore, Billerica, MA, USA). Antibodies against phospho-Akt (S473, Cat. No. ab81283), Akt (Cat. No. ab179463), phospho-FoxO1 (S256, Cat No. ab47326), FoxO1 (Cat. No. ab39670), and lamin-B1 (Cat No. ab133741) were purchased from Abcam (Cambridge, UK). Antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Cat. No. sc32233) and mouse (Cat. No. sc2005) and rabbit (Cat. No. sc2004) immunoglobulin G (IgG)-horseradish peroxidase (HRP) conjugates were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).
6
Colon Cancer Cell Line Culture and Treatment
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The human colon cancer cell lines SW48 and LIM1215 were obtained from Horizon Discovery (Cambridge, UK) and DS Pharma Biomedical (Osaka, Japan), respectively. SW48 cells were cultured in McCoy's 5A medium (Wako, Osaka, Japan) with 10% fetal bovine serum (FBS). LIM1215 cells were cultured in RPMI 1640 medium (Wako) with 10% FBS, 1 μg·mL−1 hydrocortisone (Sigma, St. Louis, MO, USA), 0.6 μg·mL−1 insulin (Thermo Fisher Scientific, Waltham, MA, USA), and 10 μm 1‐thioglycerol (Wako). Panitumumab was provided by Amgen, Inc. (Thousand Oaks, CA, USA). Cetuximab was purchased from Merck Serono (Darmstadt, Germany). FTD was purchased from Tokyo Chemical Industry (Tokyo, Japan). TPI was purchased from Biochempartner (Wuhan, China). Erlotinib was purchased from Selleck Chemicals LLC (Houston, TX, USA). U0126, LY294002, and SB203520 were purchased from Wako. Trametinib was purchased from Cayman Chemical Company (Ann Arbor, MI, USA). All antibodies used in the study were purchased from Cell Signaling Technology (Danvers, MA, USA), except anti‐glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) antibody (Merck Millipore, Billerica, MA, USA).
7
CTRP9 Signaling Pathway Analysis
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The following primary antibodies were purchased from Cell Signaling Technology (MA, USA): phospho-AMP-activated kinase (AMPK) (Thr172) antibody, AMPK antibody, phospho-Akt (Ser473) antibody, Akt antibody, phospho-endothelial nitric oxide synthase (eNOS) (Ser1177) antibody, eNOS antibody, and α-tubulin antibody. Recombinant CTRP9 was purchased from Aviscera Bioscience (CA, USA). Compound C and Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) were purchased from MilliporeSigma (MA, USA). LY294002 was purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Adenoviral vectors containing the gene for β-galactosidase (Ad-β-gal) and full-length human CTRP9 (Ad-CTRP9) were prepared as previously described (Kambara et al., 2012 (link)).
8
Angiogenesis Assay with Arnebin-1 and VEGF
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Arnebin-1 was purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan), and recombinant human VEGF was from PeproTech Inc. (Rocky Hill, NJ, USA). Growth factor-reduced Matrigel basement membrane matrix was obtained from BD Biosciences (Bedford, MA, USA). Medium 199 (M199) and fetal bovine serum (FBS) were purchased from Gibco (Carlsbad, CA, USA). LY294002, a PI3K inhibitor, was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other reagents utilized were purchased from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise specified.
9
Chick Embryo Explant Culture Assay
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EC culture was performed according to a previously described protocol (Chapman et al., 2001 (link)), with some modifications. An HH 9 chick embryo was placed on the culture gel (0.15% glucose, 0.3% agarose, 62.5 mM NaCl, ovalbumin) with the dorsal side down. Then, 100 μL of the chemical such as activators and inhibitors diluted in Tyrode’s solution (8 g NaCl, 0.2 g KCl, 0.2 g CaCl2, 0.1 g MgCl2, 0.05 g NaH2PO4, 1 g glucose, 1 g NaHCO3, up to 1 L H2O) was added dropwise. The lid of the dish was closed and left at room temperature for approximately 1 h, and the dish was incubated at 38.6°C. The following chemical solutions were used: 1% DMSO (control), 1 μM PD173074 (Santa Cruz Biotechnology, TX, USA; sc-202610), 10 μM XAV939 (Cayman Chemical, MI, USA; 13,596), 20 μM CHIR-99021 (Focus Biomolecules, PA, USA; 10–1,279), 300 μM iCRT3 (Selleck, TX, USA; S8647), and 500 μM LF3 (Selleck, TX, USA; S8474), 10 μM U73122 (Cayman Chemical, MI, USA; 70,740), 100 μM LY294002 (FUJIFILM Wako, Japan; 129–04861), 50 μM Ruxolitinib (Selleck, TX, USA; S1378), 100 μM U0126 (Chemscene, NJ, USA; CS-0173).
10
Antibody and Entry Inhibitor Effects on EV Uptake
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For treatment with antibodies, HeLa or K562 cells were incubated with anti-CD33 (15 µg/mL; Southern Biotech, Birmingham, AL, USA), anti-Tim-1 (20 µg/mL; BioLegend, San Diego, CA, USA), or anti-Tim-4 antibody (20 µg/mL; Novus Biologicals, Centennial, CO, USA) for 1 h at 37 °C. The prepared EV samples were then inoculated onto the cells in the presence of the antibodies. After incubation for 4 h at 37 °C, the inoculum was removed and the cells were maintained in MEM-1%FBS for 72 h before the luciferase assay.
For treatment with various entry inhibitors, HeLa cells prepared in the 96-well plate were incubated with Pitstop 2 (30 µM; Merck), LY294002 (5 µM; FUJIFILM Wako Pure Chemical Corporation), dynasore (80 µM; Tokyo Chemical Industry Co., Tokyo, Japan), genistein (150 µM; FUJIFILM Wako Pure Chemical Corporation), or 5-[N-ethyl-N-isopropyl] amiloride (EIPA: 100 µM; Cayman Chemical) for 30 min at 37 °C. The prepared EV samples were then inoculated onto the cells in the presence of entry inhibitors. Four hours later, the cells were rinsed with 0.5 M NaCl–0.2 M acetic acid solution and then maintained in MEM–1%FBS for 72 h before the luciferase assay.
For treatment with various entry inhibitors, HeLa cells prepared in the 96-well plate were incubated with Pitstop 2 (30 µM; Merck), LY294002 (5 µM; FUJIFILM Wako Pure Chemical Corporation), dynasore (80 µM; Tokyo Chemical Industry Co., Tokyo, Japan), genistein (150 µM; FUJIFILM Wako Pure Chemical Corporation), or 5-[N-ethyl-N-isopropyl] amiloride (EIPA: 100 µM; Cayman Chemical) for 30 min at 37 °C. The prepared EV samples were then inoculated onto the cells in the presence of entry inhibitors. Four hours later, the cells were rinsed with 0.5 M NaCl–0.2 M acetic acid solution and then maintained in MEM–1%FBS for 72 h before the luciferase assay.
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