The following were purchased from Sigma-Aldrich (Dorset, UK): Dulbecco’s Modified Eagle Medium (DMEM), Penicillin/Streptomycin, MEM non-essential amino acids 100×, 0.25% Trypsin-EDTA, L-glutamine, putrescine, spermidine, spermine. Fetal bovine serum (FBS) was from Gibco by Life Technologies (Sao Paolo, Brazil), mTOR siRNA and negative control were from Fisher Chemical (Loughborough, UK), protease and phosphatase inhibitors cocktail from Roche (Indianapolis, IN, USA), BCA protein assay kit from BioVision (Milpitas, CA, USA). RIPA lysis buffer 10× from Millipore (Burlinton, MA, USA). The following antibodies were purchased from Abcam Biotechnology (Cambridge, UK) and used at the indicated dilutions: rabbit polyclonal anti-actin antibody (ab119716, 1:7, 500 dilution), goat polyclonal anti-rabbit IgG-HRP (ab205718, 1:10,000 dilution). Additionally, mouse monoclonal anti-ODC antibody (sc-398116, 1:100 dilution), mouse monoclonal anti-phospho-4EBP1 (sc-293124, 1:200 dilution), mouse monoclonal anti-phospo-p70S6K (sc-8416, 1:300 dilution), mouse monoclonal anti- eIF4E (sc-271480, 1:300 dilution) and goat anti-mouse IgG-HRP (sc-2005, 1:2,500 dilution) antibodies were from Santa-Cruz Biotechnology (Heidelberg, Germany). Rapamycin and the class 1 dual inhibitors of PI3K and mTORC1, NVP-BEZ235, were from Cayman Chemicals (Ann Arbor, MI, USA).
Nvp bez235
Manufactured by Cayman Chemical
Sourced in United States
NVP-BEZ235 is a dual PI3K/mTOR inhibitor. It functions by inhibiting the activity of phosphoinositide 3-kinase (PI3K) and the mammalian target of rapamycin (mTOR) proteins.
Automatically generated - may contain errors
Lab products found in correlation
Rapamycin, by Merck Group (2 mentions)
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Mk 2206, by Selleck Chemicals (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
Mg132, by Merck Group (2 mentions)
Protease and phosphatase inhibitors cocktail, by Roche (1 mentions)
Rapamycin, by Cayman Chemical (1 mentions)
Bca protein assay kit, by Abcam (1 mentions)
Ripa lysis buffer 10, by Merck Group (1 mentions)
Rabbit anti actin polyclonal antibody, by Abcam (1 mentions)
Goat polyclonal anti rabbit igg hrp, by Abcam (1 mentions)
Goat anti mouse igg hrp sc 2005, by Santa Cruz Biotechnology (1 mentions)
Dulbecco s modified eagle medium dmem, by Merck Group (1 mentions)
Penicillin streptomycin, by Merck Group (1 mentions)
L glutamine, by Merck Group (1 mentions)
Doxycycline, by Thermo Fisher Scientific (1 mentions)
Epoxomicin, by Merck Group (1 mentions)
Actin antibody, by Santa Cruz Biotechnology (1 mentions)
13 protocols using nvp bez235
1
Regulation of mTOR Signaling Pathway
2
Investigating NVP-BEZ235 Cytotoxicity in Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NVP-BEZ235 was purchased from Cayman Chemical Co. (Ann Arbor, MI, USA), and dissolved in dimethylsulfoxide (DMSO). Cells were treated with indicated concentration of NVP-BEZ235 (0 nM, 50 nM, 100 nM, 200 nM, 500 nM and 1000 nM, respectively). The medium and agent were replenished every 24 h at the incubation period. DMSO was added to the medium as the vehicle control and the concentration was always kept below 0.1 %. In addition, SB216763, a GSK3β inhibitor, were purchased from TargetMol (Boston, MA, USA) and used at a concentration of 10 μM in this study.
3
Evaluating Proteasome Inhibitor Efficacy
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following chemicals were used: Torin1 (Cayman Chemical), Torin2 (Cayman Chemical), LTM (Millipore), CHX (Sigma-Aldrich), rapamycin (Sigma-Aldrich), NVP-BEZ235 (Cayman Chemical), MG-132 (EMD Millipore), clasto-lactacystin lalactone (Sigma), epoxomicin (Sigma), bortezomib (Selleckchem), and doxycycline (Fisher). The following antibodies were used: ubiquitin antibody clone FK-2 (Enzo Life Sciences), PSMA1 antibody (Sigma), EIF1AX antibody (Novus Biologicals), RPL34 antibody (Abcam), and actin antibody (Santa Cruz Biotechnology).
4
Glioma Cell Lines Treated with NVP-BEZ235
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The human U87 and U251 glioma cell lines were purchased from the Cell Bank Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). Cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Hyclone, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10 % fetal bovine serum (FBS; Gibco, Invitrogen, Carlsbad, CA, USA).
Cells were treated with 100 nmol/ml NVP-BEZ235 (Cayman Chemical, Ann Arbor, MI, USA) for 6 h for the cell proliferation trial and 6 h for the Western blot analysis. Cells treated in parallel with the same amount of dimethyl sulfoxide (DMSO) served as controls.
Cells were treated with 100 nmol/ml NVP-BEZ235 (Cayman Chemical, Ann Arbor, MI, USA) for 6 h for the cell proliferation trial and 6 h for the Western blot analysis. Cells treated in parallel with the same amount of dimethyl sulfoxide (DMSO) served as controls.
5
Preparation of Combination Chemotherapies
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We purchased the drugs from the commercial sources: paclitaxel, doxorubicin/adriamycin, 5-fluorouracil, epirubicin, cyclophosphamide, crizotinib, salinomycin, thioridazine, phenethyl isothiocyanate (PEITC), sodium valproate, sodium butyrate, 5-azacytidine, and 6-mercaptopurine (Sigma-Aldrich, St. Louis, MO), NVP-BEZ235 (Cayman Chemical, Ann Arbor, MI), and itraconazole (Selleckchem, Houston, TX). Naoto Ueno kindly provided AZD6244 (AstraZeneca, Wilmington, DE) and erlotinib (ChemieTek, Indianapolis, IN). We dissolved BEZ235, PEITC, itraconazole, crizotinib, salinomycin, doxorubicin, paclitaxel, AZD6244, and erlotinib in dimethyl sulfoxide (DMSO), thioridazine, sodium valproate, and sodium butyrate in water, 6-mercaptopurine in 0.1 M NaOH, and 5-azacytidine in dulbecco’s phosphate buffered saline. To prepare FAC (5-fluorouracil, adriamycin, cyclophosphamide) and FEC (5-fluorouracil, epirubicin, cyclophosphamide) chemotherapy combinations, drugs were measured individually and dissolved into DMSO and combined. Final concentrations of drugs were 250 nM 5-fluorouracil, 25 nM adriamycin, and 250 nM cyclophosphamide for FAC and 250 nM 5-fluorouracil, 50 nM epirubicin, and 250 nM cyclophosphamide for FEC. We added equal volume of the solvent in all dishes including the control dishes without drugs. DMSO volume was ≤0.04% of the volume of the culture medium.
6
BEAS-2B Cell Culture and Treatment
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
BEAS-2B normal human epithelial cell line was purchased from ATCC (Catalog number: CRL-9609) and cultured according to vendor instructions using BEGM kit from LONZA (Catalog number: CC-3170). Cells were cultured on 96-well black μ-plate from ibidi (Catalog Number: 89626) for imaging studies. Tanespimycin (abcam ab141433), Acetylcysteine (Cayman, 20261), Amifostine (Cayman 14398), Bortezomib (Ayman 10008822), FK-866 (Cayman 13287), Gemcitabine (Cayman 11690), Idarubicin (Cayman 14176), NVP-AUY922 (Cayman 10012698), NVP-BEZ235 (Cayman 10565), PIK-75 (Cayman 10009210), SN-38 (Cayman 15362), Tretinoin (Cayman 11017), YM-155 (Cayman 11490), Ingenol (Cayman 14031), Sulforaphane (LKT S8044), CD-437 (Sigma C5865), and Parbendazole (Sigma 1498706) were dissolved in DMSO. 1000x concentration working solution was used for downstream experimentation.
7
MYCN and NCYM Transgenic Mice Treatment
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All mice were genotyped to detect the presence of human MYCN or NCYM transgenes. After weaning, at about 30 days old, MYCN transgenic mice or MYCN/NCYM double transgenic mice were palpated for intra-abdominal tumors every day. Mice of either genotype found with palpable tumors were treated with NVP-BEZ235 (Cayman Chemical, Ann Arbor, MI, USA) (35 mg/kg in PEG300) or vehicle (PEG300, Wako) once daily for 30 days by oral gavage. All mice were monitored until euthanasia was required in accordance with the institutional animal committee.
8
Characterization of Gastric and Esophageal Cancer Cell Lines
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human gastric adenocarcinoma cell line NCI-N87 (RRID: CVCL_1603) was purchased from American Type Culture Collection (Manassas, VA). Human esophageal adenocarcinoma cell line OE19 (RRID: CVCL_1622) was purchased from European Collection of Cell Cultures (Salisbury, UK). These cell lines were confirmed by STR profiling and were Mycoplasma free. Cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, penicillin (100 U/mL), and streptomycin (100 μg/mL; Life Technologies) and were incubated at 37 °C in a humidified chamber containing 5% CO2. Tmab was provided by Chugai Pharmaceutical (Tokyo, Japan) for nonclinical investigations; 5-FU was purchased from Wako (Tokyo, Japan); CDDP was provided by YAKULT HONSHA (Tokyo, Japan). LY294002 and NVP-BEZ235 were purchased from Cayman Chemical (Ann Arbor, MA). Everolimus (RAD001) and MK2206 were purchased from Selleck Chemicals (Houston, TX).
9
Cell culture and reagent details
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Reagents included rapamycin and torin2 (both Sigma, St. Louis, MO, USA), INK-128 and NVP-Bez235 (both Cayman Chemical Company, Ann Arbor, MI, USA), erlotinib (Sequoia Research Products, Pangbourne, UK), PD153035 and bafilomycin A1 (both Tocris, Minneapolis, MN, USA). LN-308 and LNT-229 were a kind gift from Dr. N. de Tribolet (Lausanne, Switzerland). G55 cells were a kind gift from Manfred Westphal and Kathrin Lamszus (Hamburg, Germany). The genetic background of the cell lines regarding O-6-methylguanine-DNA methyltransferase (MGMT), isocitrate dehydrogenase 1 (IDH1), p53 (TP53) and PTEN are shown below (Table 1 ) [20 (link),27 (link),45 (link)]. T98G cells were obtained from ATCC (Rockville, MD, USA). Human HCT116 colon carcinoma cells were kindly provided by B. Vogelstein. MDA-MB-231 cells were a kind gift from Winfried Wels (Frankfurt, Germany). Cells were maintained at 37 °C and 5% CO2 using Dulbecco’s modified eagle medium (DMEM) supplemented with 100 IU/mL penicillin, 100 µg/mL streptomycin (Life Technologies, Karlsruhe, Germany) and 10% fetal calf serum (Biochrom KG, Berlin, Germany).
10
TORC2 Kinase Activity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To monitor the protein kinase activity of TORC2, samples of TORC2 (each derived by immuno-isolation from the equivalent of 50 µg clarified lysate, as described above) were incubated with purified MBP-Ypk1CT or MBP-Ypk1CT(6A), in kinase assay buffer in the absence or presence of the indicated amounts of GTPγS-loaded Vps21 or Ypt7, and reactions were initiated by the addition of ATP (10 µM final concentration) containing 2 µCi [γ-32P]ATP. Reactions were incubated at 30°C for 30 min unless otherwise noted, and the products were analyzed in the same manner as for the Ypk1 protein kinase assays, except SYPRO Ruby (Thermo Fisher Scientific) was used for protein staining. For specific inhibition of WT TORC2, the enzyme was incubated with QL-IX-55 (gift of Nathanael Gray, Harvard Medical School, Cambridge, MA) dissolved in DMSO or with an equivalent volume of the same solvent at 30°C for 5 min before addition of the other reaction components. For specific inhibition of the TORC2 containing Tor2(L2178A), the enzyme was incubated with NVP-BEZ235 (Cayman Chemical) dissolved in dimethyl formamide or with an equivalent volume of the same solvent.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!