Pha 665752

Manufactured by Bio-Techne

Sourced in United Kingdom, Macao

PHA-665752 is a small molecule inhibitor that targets the PI3K/Akt/mTOR signaling pathway. It functions by inhibiting the activity of PI3K and mTOR, key enzymes involved in cell growth, proliferation, and survival. PHA-665752 can be used in cell-based and in vitro assays to study the effects of PI3K/Akt/mTOR pathway modulation.

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Lab products found in correlation

Dimethyl sulfoxide (dmso), by Merck Group (3 mentions) Rapamycin, by Merck Group (2 mentions) Insulin syringe, by BD (2 mentions) Imatinib, by Bio-Techne (1 mentions) Nvp aew541, by Selleck Chemicals (1 mentions) Tae684, by Selleck Chemicals (1 mentions) Bortezomib, by LC Laboratories (1 mentions) Lapatinib, by Abcam (1 mentions) Pd173074, by Bio-Techne (1 mentions) Recombinant human hgf, by R&D Systems (1 mentions) Antibody to β actin, by Merck Group (1 mentions) Polybrene, by Merck Group (1 mentions) Puromycin, by Merck Group (1 mentions) Bez235, by Axon Medchem (1 mentions) Ceritinib, by Active Motif (1 mentions) Cabozantinib, by Active Motif (1 mentions) Lorlatinib, by Active Motif (1 mentions) Alectinib, by Active Motif (1 mentions) Azd4547, by Selleck Chemicals (1 mentions) P erk 9101, by Cell Signaling Technology (1 mentions)

8 protocols using pha 665752

Inhibitor Screening and Cell Treatment

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Imatinib (1 μM) was purchased from Tocris Bioscience. BKM-120 (5 μM) was purchased from Active Biochemical Co. dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich. NVP-AEW541 (5 μM) was purchased from Selleck. Bortezomib (50 nM) was purchased from LC Laboratories, GSK11220212 (250 nM) was provided by the Cantley lab (BIDMC). Cells in normal growth conditions, 70% confluence, were treated with inhibitors for one hour prior to lysis using DMSO as vehicle.
PHA-665752 was purchased from Tocris. Gefitinib (1 μmol/L) were obtained from American Custom Chemical. TAE-684 (100 nmol/L) was purchased from Selleck. Rapamycin (50 nmol/L) was purchased from Sigma. Cells in normal growth conditions, 70% confluence, were treated with inhibitors for 6 hour, except for Rapamycin, which was used for 16 hours prior to lysis using DMSO as vehicle.

Breitkopf S.B., Yang X., Begley M.J., Kulkarni M., Chiu Y.H., Turke A.B., Lauriol J., Yuan M., Qi J., Engelman J.A., Hong P., Kontaridis M.I., Cantley L.C., Perrimon N, & Asara J.M. (2016). A Cross-Species Study of PI3K Protein-Protein Interactions Reveals the Direct Interaction of P85 and SHP2. Scientific Reports, 6, 20471.

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Preparation and Storage of Cell Signaling Reagents

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PHA-665752 and PD-173074 were obtained from Tocris Bioscience (Bristol, UK). Lapatinib was obtained from Bio Vision (Milpitas, USA). Recombinant human HGF, recombinant human FGF1, 7, and 10 were obtained from R & D System (Minneapolis, USA). Stock solutions of PHA-665752, PD-173074 and Lapatinib were prepared in dimethyl sulfoxide and stored at −80°C until use. Stock solutions of HGF and FGFs were prepared in phosphate-buffered saline (PBS) and stored at −80°C until use.

Saito S., Morishima K., Ui T., Hoshino H., Matsubara D., Ishikawa S., Aburatani H., Fukayama M., Hosoya Y., Sata N., Lefor A.K., Yasuda Y, & Niki T. (2015). The role of HGF/MET and FGF/FGFR in fibroblast-derived growth stimulation and lapatinib-resistance of esophageal squamous cell carcinoma. BMC Cancer, 15, 82.

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Monoclonal and Polyclonal Antibodies for Protein Analysis

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Monoclonal antibody to p53 and polyclonal antibodies to EGFR, MET, and AXL were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Monoclonal antibodies to AKT1 and S6, and polyclonal antibodies to AKT, AKT2, and AKT3 were from Cell Signaling Technology (Beverly, MA, USA). Antibodies to MAPK, MDM2, and p21 were from Zymed/Invitrogen Laboratories (Invitrogen life Technologies, Carlsbad, CA, USA). All phospho-specific antibodies were from Cell Signaling Technology. Polybrene, puromycin, and antibody to β-actin were from Sigma-Aldrich (St Louis, MO, USA). Lentiviral shRNA constructs were from The RNAi Consortium (TRC, Cambridge, MA, USA), and included AXL: 5′-GCTGTGAAGACGATGAAGATT-3′; AKT1: 5′-CGCGTGACCATGAACGAGTTT-3′ (shRNA1), 5′-CGAGTTTGAGTACCTGAAGCT-3′ (shRNA2), 5′-CTATGGCGCTGAGATTGTGTC-3′ (shRNA3); and AKT2: 5′-CTTCGACTATCTCAAACTCCT-3′ (shRNA1), 5′-CAAGGTACTTCGATGATGAAT-3′ (shRNA2); and AKT3: 5′-AGAAACCTCAAGATGTGGATT-3′ (shRNA1), 5′-TGGCACACACTCTAACTGAAA-3′ (shRNA2).
Gefitinib, LY294002, U0126, GDC0941, and everolimus (RAD001) were obtained from LC Labs (Woburn, MA, USA). PHA-665752 was from Tocris Biosciences (St Louis, MO, USA). BEZ235 was from Axon MedchemBV (GZ Groningen, the Netherlands). All inhibitors were reconstituted in DMSO.

Zhou S., Liu L., Li H., Eilers G., Kuang Y., Shi S., Yan Z., Li X., Corson J.M., Meng F., Zhou H., Sheng Q., Fletcher J.A, & Ou W.B. (2014). Multipoint targeting of the PI3K/mTOR pathway in mesothelioma. British Journal of Cancer, 110(10), 2479-2488.

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Sciatic Nerve Transection and Treatment

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All surgical protocols were approved by the International Animal Care and Use Committee at Seoul National University. For sciatic nerve transection, 10-week-old male C57BL/6 mice were anesthetized with isoflurane. The sciatic nerve of the right leg was cut, and a 3-mm piece was excised. To prevent nerve reattachment, severed nerve endings were tied with 6-0 black silk suture (AILEE, Pusan, Korea). Then the incision was sutured using 5-0 silk suture (AILEE, Pusan, Korea). Sham surgery was performed by following the same procedure without severing the sciatic nerve. PHA-665752 (Tocris Bioscience, MO), a c-met inhibitor, was dissolved in DMSO (Sigma-Aldrich, MO) and administered i.p. to each mouse on a daily basis with a dose of 20 mg/kg. For i.m. injection, a 0.3-mm needle size and 0.5 mL insulin syringe (BD Biosciences, NJ) were used. The pCK or pCK-HGF-X7 plasmid expression vector was dissolved in 50 μL PBS (2 μg/μL). The injection procedure was performed by injecting the needle parallel to the tibia and then delivering the plasmid into the middle of the TA muscle.

Choi W., Lee J., Lee J., Ko K.R, & Kim S. (2018). Hepatocyte Growth Factor Regulates the miR-206-HDAC4 Cascade to Control Neurogenic Muscle Atrophy following Surgical Denervation in Mice. Molecular Therapy. Nucleic Acids, 12, 568-577.

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Muscle Injury and Regeneration Protocol

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All surgical protocols were approved by the International Animal Care and Use Committee at Seoul National University. For muscle injury, ten-week-old male C57BL/6 mice were anesthetized with isoflurane, and the TAs were injected with 50 μl CTX (Latoxan, Valence, France), diluted to 10 μM in phosphate buffered saline (PBS). Sham treatment was performed by following the same procedure except injecting TAs with PBS. PHA-665752 (Tocris Bioscience, MO), a c-met inhibitor, was dissolved in DMSO (Sigma Aldrich, MO) and i.p. administered in each mouse on a daily basis with a dose of 20 mg/kg. For i.m. injection, 0.3 mm needle size, 0.5 ml insulin syringe (BD, NJ) was used. pCK or pCK-HGF-X7 plasmid expression vector was dissolved in 50 μl PBS (2 μg/μl). The injection procedure was performed by injecting the needle parallel to the tibia and then delivering plasmid into the middle of the TA.

Choi W., Lee J., Lee J., Lee S.H, & Kim S. (2019). Hepatocyte Growth Factor Regulates Macrophage Transition to the M2 Phenotype and Promotes Murine Skeletal Muscle Regeneration. Frontiers in Physiology, 10, 914.

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Small Molecule Inhibitor Panel

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Ceritinib, alectinib, lorlatinib, cabozantinib, and zoligratinib were purchased from ActiveBiochem. Crizotinib, brigatinib, and infigratinib were purchased from Biochempartner. AZD4547 was purchased from Selleck, and PHA665752 was purchased from Tocris Bioscience.

Sakashita T., Yanagitani N., Koike S., Low S., Takagi S., Baba S., Takeuchi K., Nishio M., Fujita N, & Katayama R. (2022). Fibroblast growth factor receptor 3 overexpression mediates ALK inhibitor resistance in ALK‐rearranged non–small cell lung cancer. Cancer Science, 113(11), 3888-3900.

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Antibody Preparation and Reagents

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MUC20 antibody was prepared as described in our previous study [24 (link)]. Antibody against β-actin (A5441) was obtained from Sigma. Antibodies against MET (GTX100637), AKT (GTX121937), NFκB (GTX102090), and p-NFκB (GTX50098) were purchased from GeneTex Inc. (Irvine, CA, USA). Antibodies for immunoprecipitation of MET (#8198) and for MET pY1234/5 (#3077), p-AKT (#4060), ERK (#9102), and p-ERK (#9101) were purchased from Cell Signaling Technology, Inc. (Danvers, MA, USA). Recombinant HGF was purchased from Sigma. PHA665752, MET inhibitor, was purchased from Tocris Bioscience (Bristol, UK). SP600125, JNK inhibitor, was purchased from Selleckchem (Houston, TX, USA).

Chen S.T., Kuo T.C., Liao Y.Y., Lin M.C., Tien Y.W, & Huang M.C. (2018). Silencing of MUC20 suppresses the malignant character of pancreatic ductal adenocarcinoma cells through inhibition of the HGF/MET pathway. Oncogene, 37(46), 6041-6053.

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Combinatorial Targeting of Proliferation Pathways

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For growth curve and cell viability assays, MB cells or hNSCs were seeded in 24-well plates at the same density. Cells were treated for 72 h with mTOR (rapamycin, Sigma; Torin1, Calbiochem), FLT3 (TCS359, SelleckChem) or MET (PHA665752, Tocris) inhibitors or with inositol hexakisphosphate (IP6, Sigma) at the indicated concentrations. At specific time points or after appropriate treatment, cells were harvested, and the number of viable cells was counted with a haemocytometer and Trypan Blue staining or with CyQUANT Direct Red Cell Proliferation Assay Kit (ThermoFisher Scientific). Syenrgy/antagonist effect of the combined treatment was identified by Loewe Model with Combenefit software^{88 (link)}. Apoptosis was assessed with Caspase-3 Colorimetric Assay kit (ab39401, Abcam) following the manufacturer’s protocol. Briefly, cells treated with increasing concentrations of IP6 were lysed, and 50 µg of protein lysate was incubated with DEVD-AFC substrate and reaction buffer for 2 h at 37 °C.

Badodi S., Pomella N., Zhang X., Rosser G., Whittingham J., Niklison-Chirou M.V., Lim Y.M., Brandner S., Morrison G., Pollard S.M., Bennett C.D., Clifford S.C., Peet A., Basson M.A, & Marino S. (2021). Inositol treatment inhibits medulloblastoma through suppression of epigenetic-driven metabolic adaptation. Nature Communications, 12, 2148.

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