Wortmannin

Manufactured by Cell Signaling Technology

Sourced in United States

Wortmannin is a cell-permeable small molecule that inhibits phosphoinositide 3-kinase (PI3K) and related enzymes. It is commonly used as a research tool to study the role of PI3K signaling in cellular processes.

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62 protocols using wortmannin

Angiotensin II Signaling in APP Cells

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hAPP/Agtr1a^+/+, hAPP/Agtr1a^+/− and hAPP/Agtr1a^−/− cells were grown up to 70% confluence and starved overnight in serum-free medium prior to treatment. Starved fibroblasts were administered 100 nM Ang II (Peptide Institute) for 5, 10, 15 and 30 minutes and washed with 1 mM sodium orthovanadate before being lysed with 20 mM HEPES pH 7.0, 0.5% deoxycholic acid, 0.15 M NaCl, 0.1% SDS, 1% Nonidet P-40, 4 mM EDTA, 10 mM NaF, 10 mM Na₄P₂O₇, 2 mM sodium orthovanadate, containing a protease inhibitor cocktail (Roche). Olmesartan (1 μM, TRC), wortmannin (500 nM, Cell Signaling), perifosine (5 μM, Selleckchem) or PI3K activator (1 μg/ml, Santa Cruz Biotechnology) were added to the cells 2 h before Ang II treatment. The PI3K activator is a 1732.8 Da peptide with the sequence KKHTDDGYMPMSPGVA. This peptide binds to the SH2 domain of the PI3Kinase by the tyrosine phosphorylated version to activate the enzyme35 (link).

Liu J., Liu S., Matsumoto Y., Murakami S., Sugakawa Y., Kami A., Tanabe C., Maeda T., Michikawa M., Komano H, & Zou K. (2015). Angiotensin type 1a receptor deficiency decreases amyloid β-protein generation and ameliorates brain amyloid pathology. Scientific Reports, 5, 12059.

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Multiparameter Flow Cytometry of Immune Cells

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The following fluorochrome-labeled antibodies (Abs) specific to mouse markers and their corresponding isotype controls were purchased from eBioscience (San Diego, CA): PE-conjugated anti-IL-22 (1H8PWSR), APC-conjugated anti-IL-17A (eBio17B7), FITC- or APC-conjugated anti-IFN-γ (XMG1.2), APC-conjugated anti-TCRγδ (eBioGL3), PE-Cy7-conjugated anti-CD3 (17A2), Pacific Blue-conjugated anti-CD4 (GK1.5), PerCp-Cy5.5-conjugated anti-CD8 (53–6.7), FITC- or APC-conjugated anti-NK1.1 (PK136), FITC- or PerCp-Cy5.5-conjugated anti-CD11b (M1/70), FITC-conjugated anti-CD11c (N418), APC-conjugated anti-Gr-1 (clone: RB6-8C5), APC-conjugated anti-RORγt (B2D), Cell proliferation dye eFluor 670 and eFluor 506-conjugated fixable viability dye. Purified anti-CD16/32 (2.4G2) was purchased from BD Pharmingen (San Diego, CA). APC-Cy7-conjugated anti-CD3 (17A2) and Pacific Blue-conjugated anti-CD8 (53–6.7) were purchased from Biolegend (San Diego, CA). Recombinant murine IL-23, IL-22 and GM-CSF were from Peprotech (Rocky Hill, NJ). The all-trans RA was from Enzo Life Sciences (Farmingdale, NY). The Rapamycin, Ly294003, Wortmannin and rabbit mAb β-actin (D6A8), Phospho-p44/42 MAPK (Erk1/2), p44/42 MAPK (Erk1/2), Phospho-IκBα (Ser32) and IκBα (44D4)) were purchased from Cell Signaling Technology (Beverly, MA). Anti-S100A4 Ab (ab27957) was from Abcam (Cambridge, MA).

Jie Z., Liang Y., Yi P., Tang H., Soong L., Cong Y., Zhang K, & Sun J. (2017). Retinoic acid regulates immune responses by promoting IL-22 and modulating S100 proteins in viral hepatitis. Journal of immunology (Baltimore, Md. : 1950), 198(9), 3448-3460.

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Establishment and Manipulation of Mouse Hepatocyte Cell Lines

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Mouse hepatocyte cell lines were established from Pten‐control and Pten‐null mice.23, 24 The use of mouse embryonic fibroblast (MEF) cells has been reported before.25 AMPK plasmids and short hairpin RNA for Lkb1 (shLkb1) transfections were conducted with Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions as described.24 We plated 1 × 10⁵ cells in each well of six‐well plates 24 hours before transfection. Then, 4 μg plasmid DNA and 10 μL lipofectamine were delivered into cells. The combination of 100 pmol short hairpin (sh)RNA and 5 μL lipofectamine was used for shRNA transfection. Wortmannin, LY294002, and 5‐aminoimidazole‐4‐carboxamide ribonucleotide (AICAR) (all from Cell Signaling Technology) were used to treat the cells in vitro.

Jia C., Medina V., Liu C., He L., Qian D., Tu T., Okamoto C.T, & Stiles B.L. (2017). Crosstalk of LKB1‐regulated and PTEN‐regulated signals in liver morphogenesis and tumor development in mice. Hepatology Communications, 1(2), 153-167.

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Studying NF-κB Activation Mechanisms

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To study the action mechanism, various pharmacological inhibitors were tested on PMA-mediated NF-κB activation. Pharmacological inhibitors were used such as Wortmannin, Bay 11-7082, Genistein, GF109203X, PD98059, SB203580, SP600125, and U-73122 (Cell Signaling Technology, Danvers, MA) for the inhibition of phosphoinositide 3-kinase (PI3K), IkB-α phosphorylation, protein tyrosine kinase (PTK), protein kinase C (PKC), MEK1, SAPK2 (p38), jun N-terminal kinase (JNK), and phospholipase C (PLC), respectively.

Oh D.R., Kang H.W., Kim J.R., Kim S., Park I.K., Rhee J.H., Oh W.K, & Kim Y.R. (2014). PMA Induces Vaccine Adjuvant Activity by the Modulation of TLR Signaling Pathway. Mediators of Inflammation, 2014, 406514.

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Inhibition of Insulin Signaling in Epidermal Model

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An in vitro model of the human epidermis (LabCyte EPI-MODEL) and assay medium were obtained from J-TEC (Gamagori, Japan), and cells were cultured in an atmosphere of 95% air and 5% CO₂ at 37°C. On day 2 of culture, insulin signaling was inhibited with the insulin signaling inhibitor wortmannin (Cell Signaling) or diminished through transduction with the IR shRNA virus (25 PFU/cell MOI). Cells receiving the former treatment were administered 2 μmol/l wortmannin and cultured for 3 days, whereas those receiving the latter treatment were exposed to IR shRNA virus stock solution for 14 h, and then incubated for 3 days in assay medium. At the end of the experiment, the cells were washed with PBS (-) (Thermo Fisher Scientific), and samples were collected for gene and protein expression analysis.

Aoki M, & Murase T. (2019). Obesity-associated insulin resistance adversely affects skin function. PLoS ONE, 14(10), e0223528.

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Inhibitor Modulation of Coculture Angiogenesis

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Cocultures were seeded as described earlier in relation to EGM-2 medium containing inhibitors. The following inhibitors were added at the indicated concentrations (determined by dose–response experiments in HUVECs): 10 μM PD98059 (#9900S; Cell Signaling Technology, Danvers, MA), 10 μM U0126 (#9903; Cell Signaling Technology), 1 μM AktVIII (#124018; Merck Millipore, Darmstadt, Germany), and 0.5 μM wortmannin (#9951; Cell Signaling Technology). Stock solutions of all inhibitors were dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich), and control cultures were treated with corresponding amounts of DMSO. Cocultures were imaged and analyzed as described earlier. Assessment of statistical significance was done using a one-tailed, two-sample Student's t test assuming unequal variance in Excel v.14.1.0.

Hellesøy M, & Lorens J.B. (2015). Cellular context–mediated Akt dynamics regulates MAP kinase signaling thresholds during angiogenesis. Molecular Biology of the Cell, 26(14), 2698-2711.

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SCF-Induced CPC Survival and Migration

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Wortmannin and PD98059 were purchased from Cell Signaling Technology and used at 200 nM and 40 μM concentrations, respectively. For the Western blot analysis, CPCs were serum starved for 24 hr and pre-treated with the inhibitors for 2 hr followed by 20 min of SCF (100 ng/ml) treatment. For the cell viability and migration assays, cells were pre-treated with the indicated inhibitors for 2 hr prior to being treated with SCF.

Vajravelu B.N., Hong K.U., Al-Maqtari T., Cao P., Keith M.C., Wysoczynski M., Zhao J., Moore IV J.B, & Bolli R. (2015). C-Kit Promotes Growth and Migration of Human Cardiac Progenitor Cells via the PI3K-AKT and MEK-ERK Pathways. PLoS ONE, 10(10), e0140798.

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Organoid Culture and Manipulation

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Crypt isolation and organoid culture were performed as previously described^{14 (link)}. Serial organoid passaging was performed every six days as previously described^{15 (link)}. Mouse rLIF (50 ng/ml, Millipore), Wortmannin (1 µM, Cell Signaling), Capivasertib (1 µM, MCE), SC79 (5 µM, Sigma), Stattic (2 µM, Sigma), SB242235 (1 µM, MCE) and CHIR99021 (3 µM, Stemgent) were used for organoid treatments. The size of organoids was evaluated by quantifying the surface area of horizontal cross section of organoids acquired from multiple random non-overlapping pictures using the ImageJ software.

Wang H., Wang J., Zhao Y., Zhang X., Liu J., Zhang C., Haffty B., Verzi M., Zhang L., Gao N., Feng Z, & Hu W. (2020). LIF is essential for ISC function and protects against radiation-induced gastrointestinal syndrome. Cell Death & Disease, 11(7), 588.

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Pharmacological Modulation of Larval Behavior

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All compounds were added to the larval media 30 min before and throughout the training and testing paradigm. Cycloheximide (C4859; Sigma-Aldrich), U0126 (9903, Cell Signaling Technology), wortmannin (9951; Cell Signaling Technology), BKM120 (S2247; Selleck Chemicals), rolipram (R6520; Sigma-Aldrich), roflumilast (S2131, Selleck Chemicals), and 8-Br-cAMP (B007; BIOLOG Life Science Institute) were dissolved in 100% DMSO and administered in a final concentration of 1% DMSO. Doses of each compound were prescreened for potential effects on baseline O-bend responsiveness to visual stimuli and short-latency C-bend responsiveness to acoustic stimuli. The defined, stereotyped kinematic parameters of both larval maneuvers were also examined (Burgess and Granato, 2007a (link), 2007b (link)). Selected doses did not change baseline behavior responsiveness or kinematic performance after 30 min or 4 hr of incubation. Immunohistochemistry with anti-phospho-ERK (4377; Cell Signaling Technology) and anti-phospho-(Ser/Thr) PKA substrate (9621; Cell Signaling Technology) was performed on paraffin-embedded larval tissue after fixation in 4% paraformaldehyde, dehydration, and sectioning at 8 µMthickness in order to demonstrate the pathway specificity of the pharmacologic inhibitors (Figure S3).

Wolman M.A., de Groh E.D., McBride S.M., Jongens T.A., Granato M, & Epstein J.A. (2014). Modulation of cAMP and Ras Signaling Pathways Improves Distinct Behavioral Deficits in a Zebrafish Model of Neurofibromatosis Type 1. Cell reports, 8(5), 1265-1270.

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Characterizing HIV-1 Nef Signaling Pathways

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PBLs (5 × 10⁶ cells) were treated with recombinant myristoylated Nef protein (rNef) from the SF2 HIV-1 strain (100 ng/ml) (cat # PR-382, Jena Bioscience, Jena, Germany). Cell pellets were collected at various times after rNef treatment, washed extensively and either lysed before western blot analysis or fixed with BD Cytofix (BD Biosciences, San Jose, CA) for 20 min before flow cytometric analysis. Some PBLs were treated with the PI3K inhibitors LY294002 (0, 25 μM, 50 μM) or Wortmannin (0, 0.5 μM, 1 μM) (Cell Signaling Technology, Beverly, MA), or with the Akt inhibitor VIII (cat # sc-202048A) (0, 25, 50 μM) (Santa Cruz Biotechnology, Santa Cruz, CA) for two hours before addition of rNef (100 ng/ml) for 30 min.

Kumar A., Abbas W., Colin L., Khan K.A., Bouchat S., Varin A., Larbi A., Gatot J.S., Kabeya K., Vanhulle C., Delacourt N., Pasquereau S., Coquard L., Borch A., König R., Clumeck N., De Wit S., Rohr O., Rouzioux C., Fulop T., Van Lint C, & Herbein G. (2016). Tuning of AKT-pathway by Nef and its blockade by protease inhibitors results in limited recovery in latently HIV infected T-cell line. Scientific Reports, 6, 24090.

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