For the xenograft model, 6-week-old female nude mice housed in specific pathogen-free environments were injected s.c. with 1.0 × 106 HCT116 derivatives mixed with DMEM medium and Matrigel (vol/vol, 1:1). Treatments were initiated when the mean tumor volume in each randomized group reached around 100 mm3. NVP-BKM120 (MedChem Express) was formulated in 0.5% methylcellulose/0.5% Tween 80) at 6 mg/ml. Etoposide (Selleckchem) was formulated in Saline at 10 mg/ml. For BKM120, treatments were carried out orally, every other day, using an application volume 25 mg/kg. For Etoposide, treatments were carried out by I.P. injection with 20 mg/kg every other day. DZNep treatments were carried out by I.P. injection with 1 mg/kg body weight twice a week for 2 weeks. Animals were sacrificed when the tumor size in the vehicle control group reached 500 to 600 mm3. Tumor volumes were determined using calipers. All experimental procedures strictly complied with the IACUC guidelines.
Nvp bkm120
Manufactured by MedChemExpress
NVP-BKM120 is a potent, selective, and orally bioavailable pan-class I phosphoinositide 3-kinase (PI3K) inhibitor. It inhibits the catalytic activity of all four isoforms of PI3K (α, β, γ, and δ).
Automatically generated - may contain errors
Lab products found in correlation
Etoposide, by Selleck Chemicals (1 mentions)
Selumetinib, by MedChemExpress (1 mentions)
Celltiter glo 3d luminescent cell viability assay, by Promega (1 mentions)
Glomax multi detection system, by Promega (1 mentions)
Torin1, by Bio-Techne (1 mentions)
Rapamycin, by Cell Signaling Technology (1 mentions)
Schwann cell media scm, by ScienCell (1 mentions)
Schwann cell growth supplement scgs, by ScienCell (1 mentions)
Poly l lysine (pll), by Merck Group (1 mentions)
Annexin 5 fitc, by BD (1 mentions)
7 aminoactinomycin d 7 aad, by BD (1 mentions)
3 protocols using nvp bkm120
1
Xenograft Model for Cancer Therapeutics
2
Organoid-derived Tumor Cell Viability Assay
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KraslslG12D/wt; p53lox/lox and KrasG12D/wt; p53∆/∆ organoids were seeded in 100‐µL 10% Matrigel per 96‐well. Two, 24, and 48 hours after seeding, the organoids were lysed and CellTiter‐Glo 3D Luminescent Cell Viability Assay (Promega, Madison, WI) was performed according to the manufacturer’s protocol.
For inhibitor treatment, KrasG12D/wt; p53∆/∆; LMP_shRenilla.713 tumor cell lines established as 2D or 3D cultures from primary, organoid‐derived tumors were plated at 1,000 cells (2D cell line) and 10,000 cells (3D cell line) per 96‐well and treated with selumetinib (3.36 µM; MedChem Express, Monmouth, NJ), NVP‐BKM120 (0.25 µM; MedChem Express), or a combination of both inhibitors for 24 hours and 48 hours. At the indicated time points, cells were lysed using CellTiter‐Glo Luminescent Cell Viability Assay or CellTiter‐Glo 3D Luminescent Cell Viability Assay, and luminescence was acquired on a Glomax Multi Detection System (Promega, Madison, WI).
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KraslslG12D/wt; p53lox/lox and KrasG12D/wt; p53∆/∆ organoids were seeded in 100‐µL 10% Matrigel per 96‐well. Two, 24, and 48 hours after seeding, the organoids were lysed and CellTiter‐Glo 3D Luminescent Cell Viability Assay (Promega, Madison, WI) was performed according to the manufacturer’s protocol.
For inhibitor treatment, KrasG12D/wt; p53∆/∆; LMP_shRenilla.713 tumor cell lines established as 2D or 3D cultures from primary, organoid‐derived tumors were plated at 1,000 cells (2D cell line) and 10,000 cells (3D cell line) per 96‐well and treated with selumetinib (3.36 µM; MedChem Express, Monmouth, NJ), NVP‐BKM120 (0.25 µM; MedChem Express), or a combination of both inhibitors for 24 hours and 48 hours. At the indicated time points, cells were lysed using CellTiter‐Glo Luminescent Cell Viability Assay or CellTiter‐Glo 3D Luminescent Cell Viability Assay, and luminescence was acquired on a Glomax Multi Detection System (Promega, Madison, WI).
3
Rat Schwann Cell Primary Culture
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+ Expand
Rat primary Schwann cells (pSC) were derived from neonate rat sciatic nerves and obtained along with the Schwann cell media (SCM) and Schwann cell growth supplement (SCGS), from ScienCell Research Laboratories™ (Carlsbad, CA). Palmitic acid (PA), DHA, and poly‐L lysine were provided by Sigma‐Aldrich (St. Louis, MO). Fatty acid‐free BSA and LY294002 were purchased from EMD Biosciences (San Diego, CA). MedChem Express provided our NVP‐BKM120 (Princeton, NJ). Annexin V FITC and 7‐amino‐actinomycin D (7AAD) were purchased from BD Biosciences (San Diego, CA) and eBioscience (San Diego, CA) respectively. Finally, we obtained the antibodies to total AKT (mouse, Cat# 2920, http://scicrunch.org/resolver/AB_1147620 ) and phosphorylated AKT Ser473 (rabbit, Cat# 4060, http://scicrunch.org/resolver/AB_2315049 ) or Thr308 (rabbit, Cat# 2965, http://scicrunch.org/resolver/AB_2255933 ), and rapamycin from Cell signaling Technology (Danver, MA), the actin (mouse, Cat# A5441, http://scicrunch.org/resolver/AB_476744 ) from Sigma‐Aldrich (St Louis, MO) and Torin 1 from Tocris Bioscience (Bristol, UK). All of these antibodies are commercially available see characterization details in Table 1 .
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