Akt inhibitor 8

Manufactured by Cayman Chemical

Sourced in United States

Akt Inhibitor VIII is a selective inhibitor of the serine/threonine protein kinase Akt (also known as Protein Kinase B). It functions by blocking the activity of Akt, a key regulator of cellular processes such as cell growth, proliferation, and survival.

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Lab products found in correlation

Tgf β1, by R&D Systems (2 mentions) Rgd peptide, by Santa Cruz Biotechnology (2 mentions) Tocriscreen kinase inhibitor toolbox, by Bio-Techne (1 mentions) Rpe 1, by American Type Culture Collection (1 mentions) Hek293, by American Type Culture Collection (1 mentions) Hct116, by American Type Culture Collection (1 mentions) Caco 2, by American Type Culture Collection (1 mentions) Imr 90, by American Type Culture Collection (1 mentions) Mda mb 231, by American Type Culture Collection (1 mentions) Penicillin streptomycin, by Wisent (1 mentions) Fetal bovine serum (fbs), by Merck Group (1 mentions) Cycloheximide (chx), by Merck Group (1 mentions) Tamoxifen, by Merck Group (1 mentions) Doxycycline, by Merck Group (1 mentions) Blasticidin s hcl, by Thermo Fisher Scientific (1 mentions) Puromycin, by Merck Group (1 mentions) Doxorubicin, by Bio-Techne (1 mentions) Torin 1, by Cayman Chemical (1 mentions) Nutlin 3a, by Cayman Chemical (1 mentions) Pd098059, by Cayman Chemical (1 mentions)

7 protocols using akt inhibitor 8

TGF-β1 Signaling Pathway Inhibition

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HFF-1 cells were seeded in 96-well plates with DMEM at 10,000 cells/well. Cells were treated with control (DMEM with 2% FBS-added vehicle) or 4 ng/mL TGF-β1(R&D Systems) with or without each inhibitor, and supernatants were collected for FMOD ELISA. The following inhibitors were used: Akt inhibitor VIII (14870, Cayman Chemical) and LY294002 (440202, Calbiochem). The other inhibitors used were dispensed from a Tocriscreen Kinase Inhibitor Toolbox (3514, Tocris Bioscience).

Haga M., Iida K, & Okada M. (2024). Positive and negative feedback regulation of the TGF-β1 explains two equilibrium states in skin aging. iScience, 27(5), 109708.

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Mammalian Cell Culture Protocols

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All the cell lines (RPE1, Hela, U87, HCT116, MDAMB231, HEK 293, Caco-2, HT29, IMR90) were obtained from American Type Culture Collection (ATCC) or Coriell Institute and cultured according to standard mammalian tissue culture protocols and sterile technique. The cell lines were cultured in complete Dulbecco’s Modified Eagle Medium (DMEM) supplemented with Fetal Bovine Serum (FBS) (10%) (Sigma, #F1051) and penicillin-Streptomycin (1%) (Wisent, #450-201-EL). The following reagents were used in cell culture experiments: Tamoxifen (Sigma, #T5648), PD098059 (Cayman Chemical, #10006726), Torin1 (Cayman Chemical, #10997), Cycloheximide (Sigma, #C7698), Puromycin (Sigma, #P8833), Blasticidin S-HCl (ThermoFischer Scientific, #A1113903), Hygromycin B (BioShop Canada, #HYG002), Akt inhibitor VIII (Cayman Chemical #14870), MG132 (Cayman Chemical, #10012628), Doxycycline (Sigma, #D3447), Doxorubicin (Tocris, #2252), Nutlin3a (Cayman Chemical, #18585) and Rapamycin (LC Laboratories, #R5000).

Villot R., Poirier A., Bakan I., Boulay K., Fernández E., Devillers R., Gama-Braga L., Tribouillard L., Gagné A., Duchesne É., Caron D., Bérubé J.S., Bérubé J.C., Coulombe Y., Orain M., Gélinas Y., Gobeil S., Bossé Y., Masson J.Y., Elowe S., Bilodeau S., Manem V., Joubert P., Mallette F.A, & Laplante M. (2021). ZNF768 links oncogenic RAS to cellular senescence. Nature Communications, 12, 4841.

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Integrin-Mediated Cell Migration Assays

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Cells were pretreated with 20 μg/mL anti-α5 integrin
antibody (NKI-SAM-1), 10 μg/mL of anti-β1 integrin antibody
(P5D2), or 20 μg/mL of corresponding control isotype antibodies at on
ice for 30 min and migration and fibronectin stimulation assays were
performed.
Cells were treated with the AKT inhibitor VIII (10 μM; Cayman
Chemical Company, Ann Arbor, MI, USA) or with DMSO, and migration assays were
performed. In separate experiments, cells were cultured with the BAG (2 mM),
PPMP (20 μg/mL), or DMSO for 48 h and were then subjected to migration
and fibronectin stimulation assays. In RGD peptide blocking assay, cells were
pretreated with 100, 200, 400, 800 μM of RGD peptide (sc-201176; Santa
Cruz) or vehicle control at on ice for 30 min and fibronectin stimulation assays
were performed.

Mikami J., Tobisawa Y., Yoneyama T., Hatakeyama S., Mori K., Hashimoto Y., Koie T., Ohyama C, & Fukuda M. (2016). I‐branching N‐acetylglucosaminyltransferase regulates prostate cancer invasiveness by enhancing α5β1 integrin signaling. Cancer Science, 107(3), 359-368.

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TGF-β1 Regulation of FMOD Secretion

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We validated the model results using HFF-1 cell experiments with additional inhibitors at each time-point (four time-points, up to 48 h). Briefly, HFF-1 cells were seeded in 96-well plates with DMEM at 10,000 cells/well. The cells were treated with either control (DMEM with 0.1% DMSO supplemented with 2% FBS) or 4 ng/mL TGF-β1 (R&D Systems). Inhibitors for TGF-β1-treated samples were added at 0 h, 8 h, 24 h, and 32 h. Supernatants were collected at 48 h for quantification. Reconstitution buffer (0.1% BSA in 4 mM HCl PBS, R&D Systems) was used as vehicle. Supernatants were centrifuged (13,000 rpm, 15 min, 4°C) to remove cell debris and used for FMOD ELISA (Abcam). The following inhibitors were used: LY364947 (123-05981, Fujifilm) and Akt inhibitor VIII (14870, Cayman Chemical).

Haga M., Iida K, & Okada M. (2024). Positive and negative feedback regulation of the TGF-β1 explains two equilibrium states in skin aging. iScience, 27(5), 109708.

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Solubilization of Chemical Compounds

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4-fluoro-N’-[1-(2-pyridinyl)ethylidene]benzohydrazide (DN1) (Chembridge (Compound ID: 5325699)), U-73122 (a PLC inhibitor; Cayman Chemical), LY-294002 (a PI3K inhibitor; Sigma-Aldrich), and AKT Inhibitor VIII (Cayman Chemical) stocks were solubilized in DMSO.

Hartmann S., Nusbaum D.J., Kim K., Alameh S., Ho C.L., Cruz R.L., Levitin A., Bradley K.A, & Martchenko M. (2018). The role of a small molecule in the modulation of cell death signal transduction pathways. ACS infectious diseases, 4(12), 1746-1754.

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Integrin-Mediated Cell Migration Assay

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Cells were pretreated with 20 μg/mL anti‐α5 integrin antibody (NKI‐SAM‐1), 10 μg/mL anti‐β1 integrin antibody (P5D2), or 20 μg/mL corresponding control isotype antibodies at on ice for 30 min and migration and fibronectin stimulation assays were carried out.
Cells were treated with the AKT inhibitor VIII (10 μM; Cayman Chemical Company, Ann Arbor, MI, USA) or with DMSO, and migration assays were carried out. In separate experiments, cells were cultured with the BAG (2 mM), PPMP (20 μg/mL), or DMSO for 48 h and were then subjected to migration and fibronectin stimulation assays. In RGD peptide blocking assay, cells were pretreated with 100, 200, 400, or 800 μM RGD peptide (sc‐201176; Santa Cruz) or vehicle control on ice for 30 min and fibronectin stimulation assays were carried out.

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Multimodal Immune Profiling Protocol

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B16-F10 and LLC were obtained from ATCC. MC38 was obtained from Dario Vignali. B16^OVA (MO5) was obtained from Per Basse and Lou Falo. OVA-ex-pressing Vaccinia virus was originally generated by Yewdell and Bennink and obtained from Jonathan Powell. Most antibodies for flow cytometry were obtained from BioLegend. MitoTracker Green FM, MitoTracker Deep Red FM, tetramethylrhodamine ester (TMRE), and H2-DCFDA were obtained from ThermoFisher. VDAC antibody was obtained from Abcam. LC3B, pAktS473, pFoxo1/3a antibodies were obtained from Cell Signaling Technologies and detected after surface staining with simultaneous fixation and permabilization in 1.5% PFA madeup in 1X Permeabilization buffer (eBioscience). 2-NBD-glucose, m-divi-1, and Akt inhibitor VIII were purchased from Cayman Chemical. PGC1α antibody (H-300) was obtained from Santa Cruz Biotechnology, and was detected using the Foxp3 Fix/Perm kit (eBioscience) and Alexa Fluor 647 or Alexa Fluor 488-conjugated anti-rabbit immunoglobulin G (IgG) (Jackson Immunoresearch). Anti-PD-1 blocking antibody (J43) and its hamster IgG control were obtained from Bio-X-Cell. CellTrace Violet was purchased from eBioscience, and CFSE was purchased from BioLegend.

Scharping N.E., Menk A.V., Moreci R.S., Whetstone R.D., Dadey R.E., Watkins S.C., Ferris R.L, & Delgoffe G.M. (2016). The Tumor Microenvironment Represses T Cell Mitochondrial Biogenesis to Drive Intratumoral T Cell Metabolic Insufficiency and Dysfunction. Immunity, 45(2), 374-388.

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