C2C12 cells from the American Type Culture Collection (ATCC) were maintained in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen) which containing 10% FBS (Gibco) and 1% penicillin and streptomycin (Invitrogen) under standard cell culture conditions of 5% CO2 and 95% humidity. ICT (U-sea Biotech, Shanghai, China) was supplemented at concentrations of 5 μM, 10 μM or 20 μM. After 24 hours incubation, cells were collected and lysed for western blotting and real-time PCR analysis. In addition, C2C12 cells were incubated with 20 μM ICT for 1, 3, 5 and 7 days. CCK-8 assay (Sigma-Aldrich, Saint Louis, MO, USA) and morphology examination were performed according to the manufacturer’s instructions. The expression levels of MyoD and myogenin in C2C12 cells were determined by western blotting. In wortmannin experiments, C2C12 cells were pre-incubated with wortmannin (Cayman Chemical, Ann Arbor, MI, USA) at the concentration of 5 nM for 1 hour before the ICT treatment (20 μM). Cells cultured in the routine medium only throughout experiment served as the control.
Wortmannin
Manufactured by Cayman Chemical
Sourced in United States
Wortmannin is a chemical compound that acts as a potent and selective inhibitor of phosphoinositide 3-kinases (PI3Ks). It is commonly used as a research tool in cell biology and biochemistry studies.
Automatically generated - may contain errors
Lab products found in correlation
Ly294002, by Cayman Chemical (7 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (7 mentions)
Rapamycin, by Cayman Chemical (6 mentions)
U0126, by Cayman Chemical (6 mentions)
Bafilomycin a1, by Cayman Chemical (3 mentions)
Sb203580, by Cayman Chemical (3 mentions)
Ro 106 9920, by Santa Cruz Biotechnology (2 mentions)
Sp600125, by Cayman Chemical (2 mentions)
Phosstop phosphatase inhibitor cocktail, by Cayman Chemical (2 mentions)
Phosflow lyse fix buffer, by BD (2 mentions)
Phosflow perm buffer 3, by BD (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Anti c myc, by Cell Signaling Technology (2 mentions)
Anti phospho stat5 py694, by BD (2 mentions)
Pvdf membrane, by GE Healthcare (2 mentions)
Fk506, by Cayman Chemical (2 mentions)
Anti hdac1, by Abcam (2 mentions)
Luminata hrp substrate, by Merck Group (2 mentions)
Hrp conjugated species specific anti immunoglobulin light chain antibodies, by Jackson ImmunoResearch (2 mentions)
Anti beta tubulin, by Developmental Studies Hybridoma Bank (2 mentions)
45 protocols using wortmannin
1
Investigating ICT-mediated C2C12 Myogenesis
2
Sphingosine-1-Phosphate Signaling Pathway
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The main reagents used in this experiment include PF-543 citrate (SPHK1 inhibitor; MedChemExpress, HY-15425A, Monmouth Junction, NJ, USA), PD98059 (MEK1/2 inhibitor; MedChemExpress, HY-12028, Monmouth Junction, NJ, USA), S1P (Cayman chemical, 9002921, Ann Arbor, MI, USA), TY-52156 (S1PR3 antagonist; Cayman chemical, 19119, Ann Arbor, MI, USA), wortmannin (PI3K/Akt antagonist; Cayman chemical, 10010591, Ann Arbor, MI, USA). The specific primary antibodies include against SPHK1 (1:1000, CST, 12071S, Danvers, MA, USA), PBX1 (1:1000, CST, 4342S, Danvers, MA, USA), Phospho-Akt (Ser 473; 1:1000, CST, 4060S, Danvers, MA, USA), Akt (1:1000, CST, 4685S, Danvers, MA, USA), Phospho-p44/42 MAPK (Thr202/Tyr204; 1:1000, CST, 4370S, Danvers, MA, USA), S1PR3 (1:1000, Abcam, ab126622, Cambridge, UK), S1PR1 (1:1000, Abcam, ab23386, Cambridge, UK), CDK4 (1:1000, Santa Cruz Biotechnology, sc-23896, Dallas, TX, USA), CDK2 (1:1000, Santa Cruz Biotechnology, sc-6248, Dallas, TX, USA), CDK1/CDK2 (1:1000, Santa Cruz Biotechnology, sc-53219, Dallas, TX, USA), CyclinD1 (1:1000, Santa Cruz Biotechnology, sc-8396, Dallas, TX, USA), β-actin (1:1000, Santa Cruz Biotechnology, sc-47778, Dallas, TX, USA), goat anti-rabbit and anti-mouse horseradish peroxidases (HRPs; 1:5000, Proteintech, B900610 and B900620, Chicago, IL, USA).
3
Pharmacological Inhibitor Purchasing Protocol
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ACEA, Wortmannin, WIN 55,212-2, and Rapamycin were purchased from Cayman Chemical (USA). PTX was purchased from Sigma-Aldrich (Israel). YM-254890 was purchased from AdipoGen (USA). JD5037 and DO34 were purchased from MedChemExpress (China).
4
Inhibition of Signaling Pathways in Cell Viability Assay
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3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1), sGC inhibitor ODQ, non-competitive selective PDE inhibitor IBMX, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO, USA). The PKA inhibitor H89 were purchased from Enzo Life Sciences (Farmingdale, NY, USA). The sGC activator BAY 41-2272 and NF-κB inhibitor Ro 106-9920 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The extracellular regulated MAP kinase (ERK) inhibitor U0126, p38 MAP kinases inhibitor SB202190, c-Jun N-terminal kinases (JNK) inhibitor SP600125, HIF-1α inhibitor CAY10585, p38 activator anisomycin, and phosphatidylinositol-3-kinases (PI3K) inhibitor wortmannin were purchased from Cayman Chemical (Ann Arbor, MI, USA).
5
Integrin-mediated Signaling Pathway Analysis
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All reagents unless otherwise specified were from Sigma-Aldrich. Wortmannin, Ly294002, rottlerin, bisindolylmaleimide i, bisindolylmaleimide ix, Gö6983 were from Cayman Chemical. P11, GRGDSP, GRADSP, αvβ3 function blocking antibody (LM609) were from EMD Millipore. PP1, PP2, PP3 were from Calbiochem. LDH assay was from Promega. Phospho-MARCKS Ser152/156 (Cat#2741), MARCKS (Cat#5607), Phospho-PI3K (Cat#4228), p85 PI3K (Cat#4257), Phospho-PKCδ (Cat#2055), PKCd (Cat#9616) antibodies were from Cell Signaling Technology. FITC-PIP3 antibody (Cat#G345) was from Echelon. All secondary antibodies and isotype controls were from Life Technologies.
6
Fluorescent Staining Techniques
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All chemicals were dissolved in DMSO and were applied in solid or liquid half-strength MS-medium. 1-Naphthaleneacetic acid (NAA) was obtained from Duchefa, Brefeldin A (BFA) and wortmannin (WM) from Cayman Chemical, propidium iodide (PI) from Sigma, and FM4-64 from Invitrogen (Molecular probes).
7
AKT Phosphorylation in Adipose Progenitor Cells
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AP cells were isolated as described above23 (link), however all buffers used prior to fixation contained Roche PhosStop phosphatase inhibitor cocktail and 15 μM wortmannin (Cayman Chemical) to limit changes in pathway activation during isolation. Cells were fixed in BD Phosflow Lyse/Fix Buffer (BD Biosciences) and membranes were permeabilized in BD Phosflow Perm Buffer III (BD Biosciences) according to the manufacturer’s instructions. Cells were stained for p-Akt with the following antibodies from Cell Signaling phospho-AKT S473 PE conjugate (#5315, clone D9E, used at 1–100) and phopho-AKT T308 PE conjugate (#9088, clone C31E5E, used at 1–100), at 4°C overnight. As a control for the ability of this isolation procedure to maintain the phosphorylation status of AKT, WAT was excised, minced and treated with varying concentrations of insulin in PBS at 37°C for 10 minutes, briefly washed to remove insulin, and AP cells were then isolated from the minced tissue as described above. Phospho-AKT staining with both antibodies displayed an appropriate dose-response pattern for different treatments (See Supplementary Figure 2C–D ). For BrdU and p-AKT co-stain experiments, mice were treated for 12 hours with BrdU on the third night of HFD feeding, and then sacrificed.
8
Culturing HUVECs and HDMVECs for Histone H4 Experiments
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HUVECs and HDMVECs purchased from ATCC were cultured in EBM supplemented with EGM-2MV singleQuots (Lonza) without hydrocortisone in 0.2% gelatin-coated tissue culture plates. All experiments were performed using HUVECs of passage 6 or lower. Recombinant histone H4 was purchased from Cayman (catalog 10264) for in vitro experiments. Histone from calf thymus was purchased from MilliporeSigma (catalog 10223565001). Anti–histone H4 was from Cell Signaling Technology (catalog 2592). TLR2 inhibitor C29 was from Medchemexpress (catalog HY-100461), and TLR4 inhibitor TAK 242 was from MilliporeSigma (catalog 614316). Citrullinated histone H4 (catalog 17927), SCH 58261 (catalog 19676), PSB 603 (catalog 25637), and wortmannin (catalog 10010591) were purchased from Cayman.
9
Lace Plant Autophagy Modulation Protocol
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Lace plant (A. madagascariensis) cultures were propagated according to Gunawardena et al. (2006) . To test the effects of autophagy modulators on the formation of perforations, 40 ml septum-lidded vials were used (Sigma-Aldrich) according to Dauphinee et al. (2012) (link). Plants were grown in magenta boxes under daylight deluxe fluorescent lighting (Phillips) on 12-h dark-light cycles at an intensity of 125 μmol m−2 s−1 for approximately 4 weeks. They were then transferred to the vials and allowed to acclimate for 1 to 2 weeks. Once plants produced two to three perforated leaves, they were assigned randomly to a treatment group. Treatments were applied once to the liquid media and were dissolved in dimethyl sulfoxide (DMSO). The autophagy modulator treatments were optimized using a gradient of concentrations. The mock control treatment group received an equal volume of DMSO used for the autophagy modulator treatments. Optimal concentrations had no severe effects on leaf growth or showed signs of stress, which was observed at higher concentrations with the autophagy modulators. The optimized concentrations included 5 µM rapamycin (Enzo Scientific, BML-275), 1 µM AZD 8055 (AZD; ApexBio Technology, A8214), and 5 µM wortmannin (Cayman Chemical, 10010591).
10
Mast Cell Activation Assay
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Peptidoglycan (PGN) from S. aureus, mouse anti-human immunoglobulin E antibody (ε-chain specific) (anti-IgE) were purchased from Sigma. Human Myeloma IgE was from Merck. Pam3CSK4 was purchased from Invivogen. SP600125, SB203580, PD98059, ciclosporin and Bay11-7821 were from Tocris. Wortmannin was from Cayman. FITC-conjugated anti-human FcεRI antibody and FITC-conjugated mouse IgG2b isotype control were purchased from eBioscience. When chemicals were dissolved in DMSO, the final concentration of DMSO did not alter the normal response of LAD2 cells.
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