Byl719

Manufactured by Selleck Chemicals

Sourced in United States, Germany

BYL719 is a laboratory equipment product. It is a device designed for use in scientific research and analysis. The core function of BYL719 is to facilitate the measurement and processing of chemical or biological samples. No further details about its intended use or application can be provided while maintaining an unbiased and factual approach.

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Alpelisib (BYL719), (6 citations)

52 protocols using byl719

Establishing BYL719-resistant breast cancer cells

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The T47D human breast cancer cell line was purchased from the ATCC and cultured in RPMI1640 media supplemented with 10% fetal calf serum, 0.2 Units/ml of insulin, and 1% penicillin/streptomycin, as recommended. T47D cells were split in 1:3 to 1:4 twice a week. Cells with passage number lower than 20 were used in all experiments. To establish BYL719-resistant cell pools, T47D cells (200,000 per well in a 6-well plate) were exposed to increasing concentrations of BYL719 (Selleckchem, #S2814), starting with 1 μM and up to 30 μM, over 2 months. T47D resistant pool 1, 2 and 3 (herein RP1, RP2 and RP3) were generated and maintained in 1 μM BYL719, pulsed every 2 days. Similar procedures were used for the generation of T47D cells resistant to BKM-120 (Supplementary Fig. 5G).
For Saos2 cells, three independent plates of cells were chronically exposed to increasing concentrations of BYL719 (starting at 2 μM), or DMSO (similar v/v) for comparison. DMSO-treated cells reached confluency every 4–5 days, BYL719-treated cells remained quiescent for several weeks. However, after 6 weeks, BYL719-treated cells started to proliferate and to grow under selection.

Yip H.Y., Shin S.Y., Chee A., Ang C.S., Rossello F.J., Wong L.H., Nguyen L.K, & Papa A. (2024). Integrative modeling uncovers p21-driven drug resistance and prioritizes therapies for PIK3CA-mutant breast cancer. NPJ Precision Oncology, 8, 20.

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Comparison of PI3K Inhibitors in Mice

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BKM120, BYL719, and GDC-0032 were all purchased from Selleckchem and given via oral gavage in 100 μl. The mice were given 0.5% carboxymethyl cellulose (CMC) as vehicle control or PI3K inhibitors (BKM120: 30 mg/kg, BYL719: 30 mg/kg, and GDC-0032: 20 mg/kg) by gavage for 15 days (5 out of 7 days). The mice were euthanized after the treatment to check the transcriptional profiles of DLPs by RNA-seq analyses.

Lee Y.R., Chen M., Lee J.D., Zhang J., Lin S.Y., Fu T.M., Chen H., Ishikawa T., Chiang S.Y., Katon J., Zhang Y., Shulga Y.V., Bester A.C., Fung J., Monteleone E., Wan L., Shen C., Hsu C.H., Papa A., Clohessy J.G., Teruya-Feldstein J., Jain S., Wu H., Matesic L., Chen R.H., Wei W, & Pandolfi P.P. (2019). Reactivation of PTEN tumor suppressor for cancer treatment through inhibition of a MYC-WWP1 inhibitory pathway. Science (New York, N.Y.), 364(6441), eaau0159.

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Cell Signaling Pathway Analysis

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RPMI-1640 and McCoy's 5A medium, fetal bovine serum (FBS), penicillin and streptomycin were purchased from Lonza Milano SRL (Milan, Italy). ZSTK474, BYL719, TGX221, AS605240, CAL101, IPI145, Imatinib, Nilotinib and GZD824 were obtained from Selleck Chemicals (Houston, TX, USA). For cell viability determination, CellTiter 96 Aqueous One Solution Cell Proliferation Assay (MTS) was purchased from Promega (Milan, Italy). Annexin V/7-ADD detection kit was from Merck-Millipore (Darmstadt, Germany). Western blot antibodies for total Akt-1, Ser473 p-Akt-1 and Thr308 p-Akt-1 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), while all the other antibodies were from Cell Signaling Technology (Danvers, MA, USA), including the rabbit secondary antibody. Chloroquine, the mouse secondary antibody and the monoclonal β-Actin antibody were purchased from Sigma Aldrich (Milan, Italy). Signals were detected using ECL Plus reagent from Perkin Elmer (Boston, MA, USA).

Ultimo S., Simioni C., Martelli A.M., Zauli G., Evangelisti C., Celeghini C., McCubrey J.A., Marisi G., Ulivi P., Capitani S, & Neri L.M. (2017). PI3K isoform inhibition associated with anti Bcr-Abl drugs shows in vitro increased anti-leukemic activity in Philadelphia chromosome-positive B-acute lymphoblastic leukemia cell lines. Oncotarget, 8(14), 23213-23227.

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Glutamine Metabolism Regulation Protocols

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L-Glutamine, L-glutamine-¹³C₅, D-glucose, dimethyl α-KG and AOA were purchased from Sigma. Inhibitors LY294002 (pan-PI3K inhibitor), BYL-719 (p110α-specific inhibitor), BEZ235 (PI3K/mTOR dual-specificity inhibitor), GSK2334470 (PDK1 inhibitor), GSK690693 (AKT inhibitor), CHIR-99021 (GSK3β inhibitor), rapamycin (mTOR inhibitor), BI-D1870 (pan-RSK inhibitor), FMK (RSK2 inhibitor) and MG132 (proteasome inhibitor) were purchased from Selleck Chemicals. siRNAs for ATF4 (SI03019345 and SI04236337) and PDK1 (SI00301140 and SI00301154) were purchased from Qiagen. siRNAs for RSK2 (J-003026-10 and J-003026-12) were purchased from Dharmacon. siRNAs for USP8 (SR306014A and SR306014B) were purchased from Origene. shRNAs for GPT2 (TRCN0000035025 and TRCN0000035026) were purchased from Sigma. Antibodies used in this study are listed in Supplementary Table 1.

Hao Y., Samuels Y., Li Q., Krokowski D., Guan B.J., Wang C., Jin Z., Dong B., Cao B., Feng X., Xiang M., Xu C., Fink S., Meropol N.J., Xu Y., Conlon R.A., Markowitz S., Kinzler K.W., Velculescu V.E., Brunengraber H., Willis J.E., LaFramboise T., Hatzoglou M., Zhang G.F., Vogelstein B, & Wang Z. (2016). Oncogenic PIK3CA mutations reprogram glutamine metabolism in colorectal cancer. Nature Communications, 7, 11971.

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Inhibition of TTK and PI3K Pathways

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The inhibitors used in this study were TTKi (5 μM, MPI-0479605, Selleck) and PI3Ki (1 μM, BYL719, Selleck). All compounds were dissolved in DMSO at a concentration of 10 mM and stored at − 80 °C before use. The cells were treated with inhibitors for 3 days while the control cells were treated with DMSO only. The antibodies used in western blot were as follows: TTK (sc-376,842, 1:500 dilution, SANTA CRUZ, USA), β-actin (60008-1-lg, 1:3000 dilution, Proteintech Group, China), AKT (4685 T, 1:1000 dilution, CST, MA, USA), p-AKT (4056, 1:1000 dilution, CST, MA, USA), PI3K (4257, 1:1000 dilution, CST, MA, USA), p-PI3K (4228, 1:1000dilution, CST, MA, USA). Western blot was performed according to a previous publication [25 (link)]. The protein bands were analyzed by Image J software (NIH, USA).

Liu Y., Zhu K., Guan X., Xie S., Wang Y., Tong Y., Guo L., Zheng H, & Lu R. (2021). TTK is a potential therapeutic target for cisplatin-resistant ovarian cancer. Journal of Ovarian Research, 14, 128.

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Synergistic Effects of BYL719 and Cisplatin

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To assess drug efficacy, 2×10³ cells from each cell line were seeded in 96-well plates and treated with 0.05–10 μM of BYL719 (SelleckChem, Houston, TX) or 0–20 μM of cisplatin to determine IC₅₀. After determining the IC₅₀ and optimal concentrations, all five cell lines were subjected to treatments of 1 μM of BYL719 and escalating concentrations of cisplatin (0 to 20 μM) either separately or in combination for 48 hours and then cell proliferation was analyzed with Cell Proliferation Kit (MTT) (Roche Diagnostic GmbH, Mannheim, Germany) to determine the effect of combination therapy on PIK3CA and p53 mutants. Combination index was calculated using the method of constant ratio drug combination proposed by the Chou and Talalay (39 (link)) and the COMPUSYN software (www.combosyn.com), where CI < 1, = 1, and > 1 indicate synergism, additive effect, and antagonism, respectively. These results were obtained using a 96-well spectrophotometer with KC Junior Software (Bio-Tek Instruments, Winooski, VT).

García-Carracedo D., Cai Y., Qiu W., Saeki K., Friedman R.A., Lee A., Li Y., Goldberg E.M., Stratikopoulos E.E., Parsons R., Lu C., Efstratiadis A., Philipone E.M., Yoon A.J, & Su G.H. (2020). PIK3CA and p53 mutations promote 4NQO-initated head and neck tumor progression and metastasis in mice. Molecular cancer research : MCR, 18(6), 822-834.

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Cell Line Characterization and Compound Screening

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T47D, MCF7 cells were purchased from American Type Culture Collection (ATCC) in 2012 - 2015. They were authenticated using STR testing and tested negative for Mycoplasma contamination. EFM19, BT474, MDAMB453, HCC202, MDAMB361, HCC1419, MDAMB415, HCC1937, CAL51, BT20, HCC1954, and JIMT1 cells were purchased from Cancer Cell Line Encyclopedia (CCLE) at the Broad Institute in 2015-2016, and were authenticated using SpectroCHIPII-G384 by Sequenom's MassARRAY Analyzer Compact. All the cells were maintained in RPMI-1640 with 10% fetal bovine serum. BYL719, GDC0941, BKM120, AZD1208, GDC0032, PI-103, BX795, BX912, MK2204, GDC0068, sirolimus, everolimus, PP242 and WYE were purchased from Selleck Chemicals (Supplementary Material and Methods). Blasticidine was purchased from Life Technologies. LGH447 was obtained from Novartis.

Le X., Antony R., Razavi P., Treacy D.J., Luo F., Ghandi M., Castel P., Scaltriti M., Baselga J, & Garraway L.A. (2016). Systematic functional characterization of resistance to PI3K inhibition in breast cancer. Cancer discovery, 6(10), 1134-1147.

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Chondrocyte Maturation Pathway Inhibitors

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LY294002 (MERCK Millipore; Damstadt, Germany), NSC23766, BKM120, BYL719, and MK2206 (Selleckchem; Houston, TX, USA) were used as PI3 kinase signaling inhibitors. MG132 (A.G. Scientific; San Diego, CA, USA) was used as a proteasome inhibitor. Recombinant human BMP2 was obtained from Cellumed (Seoul, Korea) and Insulin–transferrin–selenium (ITS) supplements for chondrocyte maturation were purchased from Thermo Fisher (Waltham, MA, USA). Anti-HA polyclonal antibody was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Anti-V5 monoclonal- and anti-Myc polyclonal antibodies were obtained from Merck Millipore (Damstadt, Germany). Anti-Flag monoclonal antibody was purchased from Sigma (St. Louis, MO, USA). Anti-Type X collagen (COL 10) antibody (Cosmobio; Tokyo, Japan) was used for immunohistochemistry (IHC). Anti-GAPDH and anti-β-actin antibodies (AbFrontier; Seoul, Korea) were used as loading controls for Western blotting. Anti-Nkx3.2 antibody purchased from Abcam (Cambridge, UK) and custom-made anti-Nkx3.2 antibody was obtained from Cosmogenetech (Seoul, Korea). Horseradish peroxidase (HRP)-conjugated anti-mouse IgG and anti-rabbit IgG were purchased from Cell Signaling (Danvers, MA, USA) and Cy3-conjugated anti-rabbit IgG was purchased from Jackson ImmunoResearch Inc. (West Grove, PA, USA).

Kim J.A., Im S., Cantley L.C, & Kim D.W. (2015). Suppression of Nkx3.2 by phosphatidylinositol-3-kinase signaling regulates cartilage development by modulating chondrocyte hypertrophy. Cellular signalling, 27(12), 2389-2400.

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Detailed Protein Purification and Analysis

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General chemicals were from ThermoFisher Scientific or MilliporeSigma. Protein A-Sepharose beads were from GE Healthcare (catalog no.: GE17-0780-01). NP40, Brij35, and CHAPS detergents were from Pierce. Complete Protease Inhibitor Cocktail (EDTA-free) tablets was from MilliporeSigma (catalog no.: 11836170001). 7-methyl-GTP-Sepharose 4B beads were from GE Healthcare (catalog no.: 275025). Immobilon-P polyvinylidene difluoride (PVDF) membrane (0.45 μM) was from MilliporeSigma. Reagents for enhanced chemiluminescence were from either Alkali Scientific (Bright Star; catalog no.: XR92) or Advansta (WesternBright Sirius horseradish peroxidase [HRP] substrate). Torin1 was a gift from Dr David Sabatini. Rapamycin was from Calbiochem (catalog no.: 553210). BYL719 was from Selleck (catalog no.: 1020). MK2206 was from Selleck (catalog no.: 1078). NH₄Cl was from ThermoFisher (catalog no.: A661). cSNARF1-AM was from Invitrogen (catalog no.: C1272). Cariporide was from MilliporeSigma (catalog no.: SML1360).

Kazyken D., Lentz S.I., Wadley M, & Fingar D.C. (2023). Alkaline intracellular pH (pHi) increases PI3K activity to promote mTORC1 and mTORC2 signaling and function during growth factor limitation. The Journal of Biological Chemistry, 299(9), 105097.

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BYL719 Modulates PI3K/AKT/mTOR in MAC-BMSCs

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MAC-BMSCs were treated with BYL719 (Selleck, Houston, TX, USA) at the dose of 1.25, 2.5, or 5 μM 24 h after cell attachment. The protein expression of PI3K/AKT/mTOR pathway was detected by western blotting 2 h after treatment.

Cui H., Han G., Sun B., Fang X., Dai X., Zhou S., Mao H, & Wang B. (2020). Activating PIK3CA mutation promotes osteogenesis of bone marrow mesenchymal stem cells in macrodactyly. Cell Death & Disease, 11(7), 505.

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