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Pik 3

Manufactured by Selleck Chemicals
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Sourced in United States

PIK-III is a laboratory instrument designed for chemical and biochemical analysis. It is a versatile tool that can be used for various applications in research and development laboratories. The core function of PIK-III is to perform precise measurements and data analysis, enabling researchers to obtain accurate and reliable results.

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Lab products found in correlation

Chloroquine, by Merck Group (3 mentions) Monensin, by Merck Group (3 mentions) Ethanol, by Thermo Fisher Scientific (2 mentions) Bafa1, by Merck Group (2 mentions) Torin1, by Selleck Chemicals (2 mentions) D2650, by Merck Group (2 mentions) Cycloheximide (chx), by Merck Group (2 mentions) Bafilomycin a1, by Merck Group (2 mentions) Trypan blue, by Merck Group (2 mentions) Autophinib, by Selleck Chemicals (1 mentions) Doxycycline, by Merck Group (1 mentions) Lysotracker deep red, by Thermo Fisher Scientific (1 mentions) Dimethyl sulfoxide (dmso), by Merck Group (1 mentions) Nigericin, by Merck Group (1 mentions) Valinomycin, by Merck Group (1 mentions) Llome, by Merck Group (1 mentions) Salinomycin, by Merck Group (1 mentions) Bafa1, by Selleck Chemicals (1 mentions) Mk6 83, by Bio-Techne (1 mentions) Ml sa1, by Bio-Techne (1 mentions)

10 protocols using pik 3

1

AML Cell Line Profiling Protocol

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HL60, MOLM-14, MV4-11, OCI-AML2, OCI-AML3, U937, K562, THP1, and KASUMI AML cell lines were used (descriptions are provided in Supplemental Table 2). All AML cell lines were certified using their microsatellite identity and tested for mycoplasma contamination. Cells were cultured in RPMI (Gibco61870, Life Technologies® Saint Aubin, France) and supplemented with 10% fetal bovine serum (FBS) and 4 mM glutamine. VPS34 IN-1 was sourced from MRCC-PU Reagents (Dundee, Scotland). DMSO, chloroquine and doxycycline were obtained from Sigma-Aldrich (St. Louis, MO). Autophinib, PIK-III, Ferrostatine-1, Q-VAD-OPH, and necrostatine-1 were purchased from Selleckchem (Munich, Germany). Lysotracker deep red was obtained from Thermo Fischer Scientific (Asnières, France). l-Asparaginase was provided by Cochin hospital pharmacy Department.
Meunier G., Birsen R., Cazelles C., Belhadj M., Cantero-Aguilar L., Kosmider O., Fontenay M., Azar N., Mayeux P., Chapuis N., Tamburini J, & Bouscary D. (2020). Antileukemic activity of the VPS34-IN1 inhibitor in acute myeloid leukemia. Oncogenesis, 9(10), 94.
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2

Pharmacological Modulators of Cellular Processes

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BafA1, PIK-III, and AZD8055 were purchased from Selleckchem. ML-SA1 and MK6-83 were purchased from Tocris. Monensin, nigericin, salinomycin, valinomycin, and LLoMe were purchased from Sigma-Aldrich. C8 is available for purchase through ChemShuttle (catalog no. 187417).
Goodwin J.M., Walkup WG I.V., Hooper K., Li T., Kishi-Itakura C., Ng A., Lehmberg T., Jha A., Kommineni S., Fletcher K., Garcia-Fortanet J., Fan Y., Tang Q., Wei M., Agrawal A., Budhe S.R., Rouduri S.R., Baird D., Saunders J., Kiselar J., Chance M.R., Ballabio A., Appleton B.A., Brumell J.H., Florey O, & Murphy L.O. (2021). GABARAP sequesters the FLCN-FNIP tumor suppressor complex to couple autophagy with lysosomal biogenesis. Science Advances, 7(40), eabj2485.
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3

Lysosomal and Translational Inhibitors Protocol

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Bafilomycin A1 (Baf A1; Sigma-Aldrich B1793), a lysosomal inhibitor, was dissolved in sterile-filtered DMSO (Sigma-Aldrich D2650) as a 100 μM stock solution. Cycloheximide (Sigma-Aldrich C7698) was dissolved in sterile water as a 10 mg/mL stock solution and then passed through a 0.22-μm filter. Monensin (Sigma-Aldrich M5273) was dissolved in ethanol (Thermo Fisher Scientific) as a 50 mg/mL (72.2 mM) stock solution. PIK-III (Selleck Chemicals S7683), a PI3KC3 (VPS34)-specific inhibitor40 , was dissolved in sterile-filtered DMSO as a 4 mM stock solution. Torin 1 (Selleck Chemicals S2827), an mTOR inhibitor41 , was dissolved in sterile-filtered DMSO as a 1 mM stock solution. All stock solutions were stored in small aliquots at −20°C. For control groups of chemical treatment, equivalent volume of according chemical-free sterile solvent was added into culture media.
Dong X., Yang Y., Zou Z., Zhao Y., Ci B., Zhong L., Bhave M., Wang L., Kuo Y.C., Zang X., Zhong R., Aguilera E.R., Richardson R.B., Simonetti B., Schoggins J.W., Pfeiffer J.K., Yu L., Zhang X., Xie Y., Schmid S.L., Xiao G., Gleeson P.A., Ktistakis N.T., Cullen P.J., Xavier R.J, & Levine B. (2020). Sorting Nexin 5 Mediates Virus-Induced Autophagy and Immunity. Nature, 589(7842), 456-461.
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4

Protein Analysis and Transfection Assays

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The DC Protein Assay (500-0119) and Clarity Western ECL Substrate (170-5061) were from BioRad. The Effectene Transfection Reagent (301427) was from Qiagen. The Lipofectamine RNAiMAX Reagent (100014472) was from Thermo Fisher Scientific. ProLong Diamond antifade mountant without (P36970) or with (P36971) DAPI (4′,6-diamidino-2-phenylindole) was from Thermo Fisher Scientific. BafA1 (11038) and MRT68921 (1990520000040) were from Cayman Chem. PIK-III was from Selleckchem (S7683). Bort (B-1408) was from LC Laboratories. Janelia Fluor 646 HaloTag Ligand (GA1120) was from Promega.
Plasmids used and sources are given in Table 1. Antibodies used along with hosts and manufacturers appear in Table 2.
Kennedy A., Ren H.Y., Madden V.J, & Cyr D.M. (2022). Lysosome docking to WIPI1 rings and ER-connected phagophores occurs during DNAJB12- and GABARAP-dependent selective autophagy of misfolded P23H-rhodopsin. Molecular Biology of the Cell, 33(9), ar84.
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5

Lysosomal and Translational Inhibitors Protocol

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Bafilomycin A1 (Baf A1; Sigma-Aldrich B1793), a lysosomal inhibitor, was dissolved in sterile-filtered DMSO (Sigma-Aldrich D2650) as a 100 μM stock solution. Cycloheximide (Sigma-Aldrich C7698) was dissolved in sterile water as a 10 mg/mL stock solution and then passed through a 0.22-μm filter. Monensin (Sigma-Aldrich M5273) was dissolved in ethanol (Thermo Fisher Scientific) as a 50 mg/mL (72.2 mM) stock solution. PIK-III (Selleck Chemicals S7683), a PI3KC3 (VPS34)-specific inhibitor40 , was dissolved in sterile-filtered DMSO as a 4 mM stock solution. Torin 1 (Selleck Chemicals S2827), an mTOR inhibitor41 , was dissolved in sterile-filtered DMSO as a 1 mM stock solution. All stock solutions were stored in small aliquots at −20°C. For control groups of chemical treatment, equivalent volume of according chemical-free sterile solvent was added into culture media.
Dong X., Yang Y., Zou Z., Zhao Y., Ci B., Zhong L., Bhave M., Wang L., Kuo Y.C., Zang X., Zhong R., Aguilera E.R., Richardson R.B., Simonetti B., Schoggins J.W., Pfeiffer J.K., Yu L., Zhang X., Xie Y., Schmid S.L., Xiao G., Gleeson P.A., Ktistakis N.T., Cullen P.J., Xavier R.J, & Levine B. (2020). Sorting Nexin 5 Mediates Virus-Induced Autophagy and Immunity. Nature, 589(7842), 456-461.
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6

Modulation of Autophagy Pathways

Cited 1 time
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Bafilomycin A1 (Sigma-Aldrich, B1793) (Baf A1) was dissolved in sterile-filtered DMSO (Sigma-Aldrich, D2650) as 100 µM stock solutions and stored in small aliquots at −20°C. Chloroquine (Sigma-Aldrich, C6628) was freshly dissolved in phosphate-buffered saline (PBS; Sigma-Aldrich, D8537). PIK-III (Selleck Chemicals, S7683) was dissolved in DMSO as 4 mM stock solutions and stored in small aliquots at −20°C. Tat-BECN1 (YGRKKRRQRRR-GG-VWNATFHIWHD, active) and Tat-BECN1F270S, (YGRKKRRQRRR-GG-VSNATFHIWHD, inactive) peptides were synthesized by the University of Texas Southwestern Medical Center (UTSW) Protein Chemistry Technology Core and purified to >95% by HPLC (confirmed by mass spectrometry) [13 (link)]. Peptides were dissolved in PBS and stored at −80°C.
Zhao Y., Zou Z., Sun D., Li Y., Sinha S.C., Yu L., Bennett L, & Levine B. (2020). GLIPR2 is a negative regulator of autophagy and the BECN1-ATG14-containing phosphatidylinositol 3-kinase complex. Autophagy, 17(10), 2891-2904.
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7

Autophagy Assays in Cell Lines

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RPMI 1640 medium, fetal calf serum (FCS), phosphate buffered saline (PBS), were from Invitrogen. Trypan blue and the antibody against LC3 were from Sigma (St Quentin Fallavier, France). The following antibodies : ATG7 was from Cell Signalling (Danvers, USA), and Hsp60 was from Santa Cruz (Bergheimer, Germany). Annexin-V-APC was from Beckman coulter (Villepinte, France). Ki-67 antibody, control isotype and anti-Abl were purchased from BD (Becton Dickinson, France). PIK-III and SBI-0206965 were purchased from Selleck chemicals (Houston, USA).
Ianniciello A., Dumas P.Y., Drullion C., Guitart A., Villacreces A., Peytour Y., Chevaleyre J., Brunet de la Grange P., Vigon I., Desplat V., Priault M., Sbarba P.D., Ivanovic Z., Mahon F.X, & Pasquet J.M. (2017). Chronic myeloid leukemia progenitor cells require autophagy when leaving hypoxia-induced quiescence. Oncotarget, 8(57), 96984-96992.
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8

Inhibitors of Autophagy and Actin Dynamics

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Torin1 (Tocris Bioscience, 4247) was used at a final concentration of 50 nM and Rapamycin (Calbiochem, 553210) at a final concentration of 20 nM. Phalloidin (Thermo Fisher Scientific, A12379) was used according to the manufacturers’ recommendations. Latrunculin A was purchased from Tocris and used at a final concentration of 0.1 μM. PIK-III (Selleck Chemicals)31 (link) was used at a final concentration of 5 μM, Concanamycin A (Cayman Chemical Company) at 100 nM, and Chloroquine (Sigma-Aldrich) at 100 μM or 20 μM, as specified in the figure legends.
Mutvei A.P., Nagiec M.J., Hamann J.C., Kim S.G., Vincent C.T, & Blenis J. (2020). Rap1-GTPases control mTORC1 activity by coordinating lysosome organization with amino acid availability. Nature Communications, 11, 1416.
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9

In Vitro CDDP, PIK-III, and SAR405 Protocol

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CDDP for in vitro experiments was purchased from Teva. PIK-III (#S7683) and SAR405 (#S7682) were purchased from Selleckchem. Bafilomycin A1 was purchased from Sigma-Aldrich (B1793–2UG).
Vianello C., Cocetta V., Catanzaro D., Dorn GW I.I., De Milito A., Rizzolio F., Canzonieri V., Cecchin E., Roncato R., Toffoli G., Quagliariello V., Di Mauro A., Losito S., Maurea N., Scaffa C., Sales G., Scorrano L., Giacomello M, & Montopoli M. (2022). Cisplatin resistance can be curtailed by blunting Bnip3-mediated mitochondrial autophagy. Cell Death & Disease, 13(4), 398.
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10

Apoptosis Evaluation in Cell Lines

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The αMEM medium, fetal calf serum (FCS) and phosphate buffered saline (PBS) were from Invitrogen. The trypan blue and the anti-LC3 antibody were from Sigma (St Quentin Fallavier, France). The annexin-V-APC was from Biolegend (France). The PIK-III was purchased from Selleck Chemicals (Houston, TX, USA). Cell apoptosis was assessed using an APC-conjugated annexin V labeling detection kit coupled to flow cytometry and BDFACSDIVATM software Becton Dickinson, Le Pont de Claix, France. The murine G-CSF was from Peprotech (Neuilly, France) and AMD3100 from SantaCruz (Heidelberg, Germany). Gilteritinib and SAR-405 were purchased from MedChemTronica (Sollentuna, Sweden).
Dupont M., Huart M., Lauvinerie C., Bidet A., Guitart A.V., Villacreces A., Vigon I., Desplat V., El Habhab A., Pigneux A., Ivanovic Z., Brunet De la Grange P., Dumas P.Y, & Pasquet J.M. (2022). Autophagy Targeting and Hematological Mobilization in FLT3-ITD Acute Myeloid Leukemia Decrease Repopulating Capacity and Relapse by Inducing Apoptosis of Committed Leukemic Cells. Cancers, 14(2), 453.
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