Nvp bez235

Manufactured by Merck Group

Sourced in United States

NVP-BEZ235 is a dual PI3K/mTOR inhibitor that targets the PI3K and mTOR signaling pathways. It is a small molecule compound that has been used in research applications to study the role of PI3K and mTOR in cellular processes.

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5 protocols using nvp bez235

Glioma Cell Line Propagation and Inhibition

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All cell lines used in this study were provided by the American Type Culture Collection (Manassas, VA, USA). U87MG cells were propagated in MEM (Minimal Essential Medium, Thermo Fisher, Waltham, MA). DBTRG cells were propagated in RPMI (Roswell Park Memorial Institute 1640 Medium, Thermo Fisher, Waltham, MA). LN-18, U118 MG, and LN-229 cells were propagated in DMEM (Dulbecco’s Modified Eagle Medium, Thermo Fisher, Waltham, MA, USA). Cell lines were grown in standard propagating conditions supplemented with 10% fetal bovine serum (FBS, Thermo Fisher, Waltham, MA), antibiotic (Anti-Anti, Gibco cat: 15240096, Thermo Fisher, Waltham, MA), and 5% CO₂ at 37 °C. The dual PI3K-mTOR pathway inhibitor NVP-BEZ235 and FOXO1 inhibitor AS1842856 were acquired from Sigma-Aldrich (Saint Louis, MO, USA) and Calbiochem (Danvers, MA, USA), respectively. NVP-BEZ235 and AS1842856 were utilized in final concentrations at 50 nM (or 1 μM) and 200 nM, respectively. Control samples were treated with DMSO.

Udawant S., Litif C., Lopez A., Gunn B., Schuenzel E, & Keniry M. (2021). PI3K Pathway Inhibition with NVP-BEZ235 Hinders Glycolytic Metabolism in Glioblastoma Multiforme Cells. Cells, 10(11), 3065.

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Signaling Pathway Regulation in Cells

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Fluocinolone acetonide (FA), croton oil (CO), Wortmannin (WM), AZD8055 (AZD), NVP-BEZ235 (NVP), MK-2206 (MK), and 5-bromo-2′-deoxyuridine (BrdU), were from Sigma Aldrich (St. Louis, MO), LY294002 (LY) was from LC Laboratories (Woburn, MA). TNF-α and cytoplasmic and nuclear fractionation kit were from Thermo Fisher Scientific (Waltham, MA).
We used antibodies to GR (sc-8992, RRID:AB_2155784), RelA/p65 (sc-8008, RRID:AB_628017), phospho-RelA/p65 (Ser536) (sc-136548, RRID:AB_10610391), IκB (sc-1643, RRID:AB_627772), phospho-IκB (Ser32) (sc-8404, RRID:AB_627773), FKBP51 (sc-271547, RRID:AB_10649040) (Santa Cruz Biotechnology, Dallas, TX); GAPDH (G9545, RRID:AB_796208, Sigma Aldrich); phospho-GR (Ser211) (4161, RRID:AB_2155797), phospho-rpS6 (Ser235/236) (4856S, RRID:AB_2181037), phospho-4E-BP1 (Thr37/46) (9459L, RRID:AB_2262165), phospho-P70S6K (Thr389) (9234, RRID:AB_2269803), phospho-AKT (Thr308 and Ser473) (9266S, RRID:AB_659801 and 9271, RRID:AB_329825), lamin B (12586, RRID:AB_2650517) and tubulin (2148, RRID:AB_2288042) (Cell Signaling, San Jose, CA), REDD1 (10638-1-AP, RRID,AB_2245711, Proteintech Group, Rosemont, IL).

Agarwal S., Mirzoeva S., Readhead B., Dudley J.T, & Budunova I. (2019). PI3K inhibitors protect against glucocorticoid-induced skin atrophy. EBioMedicine, 41, 526-537.

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Inducing Primary Cilia Formation and Testing Inhibitors

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The image-based screen that identified NVP-BEZ235 has been previously described in^{28 (link)}. All siRNA’s (siControl, siVHL, siAKT) were obtained as lyophilized powder from Dharmacon (GE Healthcare, Lafayette, CO, USA) and resuspended in 250µL of 1X siRNA buffer (Dharmacon). Cells at 70% confluence were transfected with siC, siVHL, and siAKT at 30 nm each and serum starved for 48 h to induce the formation of primary cilia. NVP-BEZ235 (Dactolisib), MLN8237 (Alisertib), MK2206 and LY294002 were obtained from Selleck Chem (Houston, TX, USA). MK2206 and LY294002 were used at final concentrations of 2 µM and 10 µM, respectively. Rapamycin (Sigma-Aldrich St.Louis, MO, USA) was used at a final concentration of 200 nM. In vitro, NVP-BEZ235 was used at 0.1 µM, 0.3 µM and 1.0 µM (solubilized in DMSO) for 48 h in serum free media. DMSO was used as a vehicle control.

Chowdhury P., Perera D., Powell R.T., Talley T., Tripathi D.N., Park Y.S., Mancini M.A., Davies P., Stephan C., Coarfa C, & Dere R. (2021). Therapeutically actionable signaling node to rescue AURKA driven loss of primary cilia in VHL-deficient cells. Scientific Reports, 11, 10461.

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Cell Culture Conditions and Treatments

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Cell lines were obtained from ATCC (American Type Culture Collection, Manassas, VA) and grown under standard conditions (5% CO₂, 10% FBS (fetal bovine serum), with 5% antifungal/antibacterial). Cell lines were tested for Mycoplasma using the MycoAlert Mycoplasma Detection Kit (Lonza, Basel Switzerland, cat: LT07–218); all experiments were done with mycoplasma negative cells. U87MG cells were propagated in MEM (Minimal Essential Medium). BT549 and DBTRG cells were propagated in RPMI (Roswell Park Memorial Institute 1640 Medium). HEK 293, LN18, U118MG, A172, and LN229 cells were propagated in DMEM (Dulbecco’s Modified Eagle Medium). NVP-BEZ235 was purchased from Sigma (Saint Louis, MO), and utilized at a final concentration of 50 nM in indicated experiments. Two sets of experiments utilized a higher dose of NVP-BEZ235. LN229 cells were treated with 1 μM NVP-BEZ235 and data from Figs. S3A–B were generated using 1 μM NVP-BEZ235 treatment. UO126 was purchased from Selleck Chemicals (Houston, TX) and used at 10 μM final concentration. Cells were plated at a density of 2700 cells per ml and were treated for 5 days with NVP-BEZ235 (unless otherwise stated). AS1842856 was purchased from Calbiochem (Danvers, MA) and utilized at 200 nM final concentration.

Martinez E., Vazquez N., Lopez A., Fanniel V., Sanchez L., Marks R., Hinojosa L., Cuello V., Cuevas M., Rodriguez A., Tomson C., Salinas A., Abad M., Holguin M., Garza N., Arenas A., Abraham K., Maldonado L., Rojas V., Basdeo A., Schuenzel E., Persans M., Innis-Whitehouse W, & Keniry M. (2020). The PI3K pathway impacts stem gene expression in a set of glioblastoma cell lines. Journal of cancer research and clinical oncology, 146(3), 593-604.

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Evaluating Chemosensitivity in Leukemia Cells

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Cell culture and treatment. The AML cell lines HL-60 (FAB M2) and KG-1 (erythroleukemia-FAB M6) were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Cells were maintained in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) (both from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA), 1% antibiotic-antimycotic mixture (Thermo Fisher Scientific, Inc.) in a humidified, 37˚C and 5% CO 2 atmosphere. Medium was half-refreshed every 3 days.
Doxor ubicin and dual pan-PI3K /mTOR kinase inhibitor dactolisib (NVP-BEZ235) were purchased from (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). For the evaluation of chemosensitivity to doxorubicin, a range concentration from 0.5 to 100 µM was incubated with KG-1 or HL-60 cells for 24 h, respectively. For inhibition of the PI3K/AKT/mTOR pathway, KG-1 or HL-60 cells were pretreated with 0.5 Μm NVP-BEZ235 for 6 h.

Yang Y., Dai W., Sun Y, & Zhao Z. (2019). Long non‑coding RNA linc00239 promotes malignant behaviors and chemoresistance against doxorubicin partially via activation of the PI3K/Akt/mTOR pathway in acute myeloid leukaemia cells. Oncology reports, 41(4).

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