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Pp242

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PP242 is a selective and potent inhibitor of the mammalian target of rapamycin complex 1 (mTORC1). It functions by blocking the kinase activity of mTOR, a critical regulator of cell growth, proliferation, and metabolism.

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Lab products found in correlation

Rapamycin, by Merck Group (4 mentions) Monensin, by Merck Group (3 mentions) Torin1, by Bio-Techne (3 mentions) Rapamycin, by Bio-Techne (2 mentions) Puromycin, by Thermo Fisher Scientific (2 mentions) Hygromycin, by Thermo Fisher Scientific (2 mentions) Doxycycline, by Merck Group (2 mentions) Ku006, by Bio-Techne (2 mentions) Wy354, by Bio-Techne (2 mentions) Mln0128, by Bio-Techne (2 mentions) Rad001, by Bio-Techne (2 mentions) Azd8055, by AstraZeneca (2 mentions) Phorbol 12 myristate 13 acetate pma, by Merck Group (2 mentions) Everolimus, by Merck Group (2 mentions) α β catenin, by BD (2 mentions) Chir99021, by Bio-Techne (2 mentions) α p4ebp, by Cell Signaling Technology (2 mentions) α 4ebp, by Cell Signaling Technology (2 mentions) α ps6, by Cell Signaling Technology (2 mentions) Rabbit α gsk 3, by Santa Cruz Biotechnology (2 mentions)

17 protocols using pp242

1

Autophagy Protein Detection and Inhibition

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The following primary antibodies were used at the indicated dilutions: anti-LC3A/B (1:1000 for western blotting and 1:100 for immunofluorescence; Cell Signaling Technology, 4108), anti-LC3A (1:100 for immunohistofluorescence; Abgent, 1805a), anti-ATG12 (1:100; Cell Signaling Technology, 2010), anti-ATG13 (1:1000; Cell Signaling Technology, 6940), anti-ATG16L1 (1:1000; Cell Signaling Technology, 8089), anti-human LAMP1 and anti-mouse LAMP1 (1:100; Becton Dickinson, 555798 and 553792), anti-WIPI2 (1:100; Bio-Rad, MCA5780GA), anti-CDSN/corneodesmosin (R&D Systems, AF5725), anti-GAPDH (1:2000; Santa Cruz Biotechnology, sc-25778), and anti-ATP6V0D1 (1:50; ABCAM, ab56441). The following inhibitors and reagents were used: Amiodarone hydrochloride (Sigma, A8423), bafilomycin A1 (Tocris Biosciences, 1334), betahistine dihydrochloride (Sigma, B4638), chloroquine (Sigma, C6628), CCCP (Sigma, C2759), HBSS (Gibco, 14025–092), hydroxychloroquine (Sigma, H0915), lidocaine (Sigma, L7757), lidocaine hydrochloride monohydrate (Sigma, L5647), monensin (Sigma, M5273), NH4Cl (Sigma, 213330), nigericin (Sigma, N7143), PP242 (Tocris, 4257), procainamide hydrochloride (Sigma, P9391). Mito-TMRE (abcam, 113852), PIK3C3/VPS34 inhibitor (IN-1) was kindly provided by Dr I. Ganley, University of Dundee.
Jacquin E., Leclerc-Mercier S., Judon C., Blanchard E., Fraitag S, & Florey O. (2017). Pharmacological modulators of autophagy activate a parallel noncanonical pathway driving unconventional LC3 lipidation. Autophagy, 13(5), 854-867.
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2

Characterizing eIF4E-eIF4G and 4EBP1 Interactions

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All eIF4E, eIF4G (including the Y624A, L629A and L630A binding mutant) and 4EBP1 mutant cDNAs were synthetized and obtained from IDT (Integrated DNA Technologies). eIF4E and eIF4G604–646 were cloned into NanoBit plasmids using the NanoBit PPI starter system (Promega) using XhoI/EcoRI and NheI/EcoRI cloning sites respectively. 4EBP1 mutants were cloned into pCDNA3.1 vector DNA (Thermo Fisher Scientific) harbouring a C-terminal 3× FLAG tag via NheI/BamHI sites to allow mammalian cell overexpression. For bacterial expression, 4EBP1 mutants were cloned into pGEX6P1 using BamHI/EagI cloning sites. GFP and v-Myc coding sequence residing in a pCMV6 mammalian expression vector were obtained from Origene. The bicistronic luciferase reporter construct pcDNA3-rLuc-polIRES-fLuc was purchased from Addgene. PP242, Torin1, Rapamycin, 4EGi-1 and 4E1RCat were purchased from Tocris Bioscience, whilst all other chemicals unless otherwise stated were purchased from Selleck Chemicals. siRNAs targeting either 4EBP1 (ON-TargetPlus Human EI4EBP1, J-003005-12-0005) or non-targeting control (ON-TargetPlus Non-targeting pool, D-001810-10-05) were purchased from DHARMACON.
Frosi Y., Usher R., Lian D.T., Lane D.P, & Brown C.J. (2019). Monitoring flux in signalling pathways through measurements of 4EBP1-mediated eIF4F complex assembly. BMC Biology, 17, 40.
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3

Cell Treatments with Pharmacological Compounds

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Cells were treated with the following drugs at concentrations and times as indicated in the figure legends: Cycloheximide (Sigma-Aldrich, C7698), PP242 (Tocris 4257), Thapsigargin (Cayman Chemical Company 10522), Bafilomycin A (Sigma-Aldrich, B1793), Rucaparib (Selleck Chemicals, S1098), Olaparib (Cellagen Technologies, C2228-5s) BMN637 was a gift from Alan Ashworth43 (link), is commercially available from Selleck Chemicals (S7048), menadione (Sigma-Aldrich, M5625), N-acetyl cysteine (Sigma-Aldrich, A7250).
Goldsmith J., Marsh T., Asthana S., Leidal A.M., Suresh D., Olshen A, & Debnath J. (2020). Ribosome profiling reveals a functional role for autophagy in mRNA translational control. Communications Biology, 3, 388.
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4

In Vitro T-Reg Cell Suppression Assay

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For analysis of Treg-cell suppression in vitro, CD4+CD25hi Treg cells or CD4+Foxp3-YFP+ Treg cells, isolated from the lymphoid organs of the respective Cd4Cre- or Foxp3Cre-expressing mice, were co-cultured with naive CD4+ T cells and irradiated splenocytes as antigen presenting cells as previously described30 (link). For suppression assays using in vitro activated Treg cells, CD25hi Treg cells were sorted from the lymphoid organs of C57BL/6 mice, resuspended in complete Click’s medium containing IL-2 (200 U ml−1), and activated using anti-CD3 (10 μg ml−1) and anti-CD28 (10 μg ml−1) antibodies for 3 days in the presence of PP242 (500 nM, Tocris Bioscience) or vehicle control. The live cells were then isolated using Lymphocyte Separation medium and co-cultured with naive CD4+ T cells and irradiated splenocytes for 3 days, and the incorporation of [3H]-thymidine was assessed as described30 (link).
Chapman N.M., Zeng H., Nguyen T.L., Wang Y., Vogel P., Dhungana Y., Liu X., Neale G., Locasale J.W, & Chi H. (2018). mTOR coordinates transcriptional programs and mitochondrial metabolism of activated Treg subsets to protect tissue homeostasis. Nature Communications, 9, 2095.
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5

Breast Cancer Cell Line Maintenance Protocol

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All cell lines were obtained from the American Type Culture Collection (ATCC). MCF-7 and MDA-MB-468 (ATCC catalog number: HTB-22 and HTB-132 respectively) breast cancer cell lines were maintained in a 1:1 mixture of DME: F12 medium supplemented with 4 mM glutamine, 100 units/mL each of penicillin and streptomycin, and 10% heat-inactivated fetal bovine serum (FBS) and incubated at 37°C in 5% CO2. The MDA-MB-468 inducible expression cells were maintained in the same medium with addition of 50 µg/ml hygromycin and 0.2 µg/mL Puromycin. The HEK-293 cells (ATCC catalog number: CRL-1573) were maintained in DMEM medium with glutamine, antibiotics and 10% FBS. The cell lines resulted negative for mycoplasma contamination. AZD8055 was obtained from AstraZeneca Pharmaceuticals, rapamycin was purchased from EMD Bioscience. RAD001, KU006, WY354, PP242, MLN0128 were purchased from Tocris. Doxycycline was purchased from Sigma Aldrich. Puromycin and hygromycin stock solution were purchased from Invitrogen. Drugs were dissolved in DMSO to yield 10 mM stock and stored at −20°C.
Rodrik-Outmezguine V.S., Okaniwa M., Yao Z., Novotny C.J., McWhirter C., Banaji A., Won H., Wong W., Berger M., de Stanchina E., Barratt D.G., Cosulich S., Klinowska T., Rosen N, & Shokat K.M. (2016). Overcoming mTOR Resistance Mutations with a New Generation mTOR Inhibitor. Nature, 534(7606), 272-276.
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6

Jurkat and Primary T Cell Culture

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Jurkat (ATCC, Cat # TIB-152) cells were grown in RPMI-1640 with 2mM L-glutamine, 10mM HEPES and 1mM sodium pyruvate; primary T cells were grown in RPMI-1640 with 2mM L-glutamine. All media contained 10% heat-inactivated FBS, penicillin and streptomycin. Primary naïve (CD4+CD45RA+CD45RO-) and memory (CD4+CD45RA- CD45RO+) T cells were isolated from peripheral blood collected from healthy donors ages 18–30 years. Briefly, peripheral blood mononuclear cells were enriched by Ficoll- histopaque (Sigma-Aldrich) density gradient centrifugation, and T cells were purified from these cells by negative selection using magnetic beads (Miltenyi Biotec). Cells were stimulated with anti-CD3/CD28 beads (Life Technologies) using manufacturer’s instructions for 48 h. All other cell lines were obtained from ATCC and grown following the recommended culture methods. All cells were grown at 37°C in a 5% CO2 humidified chamber. Rapamycin (Sigma-Aldrich) and PP242 (Tocris) were reconstituted in DMSO. Rapamycin was used at 100 nM unless otherwise noted. Anti-CD95 antibody (Millipore) was used at 0.1ug/ml.
Podshivalova K., Wang E.A., Hart T, & Salomon D.R. (2018). Expression of the miR-150 tumor suppressor is restored by and synergizes with rapamycin in a human leukemia T-cell line. Leukemia research, 74, 1-9.
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7

Modulation of Cellular Signaling Pathways

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Cells were treated with mTOR inhibitors- Rapamycin (RAP) (10 μM) (Calbiochem) or Temisirolimus (TEM) (5 μM) (Santacruz biotechnology) or Torin-1 (TOR) (100 nM) (Tocris) or PP242 (100 nM) (Tocris) and cytokines- Tumor Necrosis Factor-α (TNFα) (10 ng/ml) (Peprotech), Interleukin1β (IL1β) (10 ng/ml) (Peprotech) and tumor promoting compound Phorbol 12- myristate 13-acetate (PMA) (100 ng/ml) (Sigma Aldrich). Staurosporin (STS) (50 nM) and UCN01 (500 nM) (Sigma Aldrich) are pan-inhibitors of PKC kinases. Amino salicylic acid (ASA) (20 μM) and BAY11 (10 μM) (Sigma Aldrich) are specific NFκB inhibitors. Experiments were performed on cells treated in complete medium except for gelatinolytic zymography, invasion and migration assays where serum-free medium was used.
Chandrika G., Natesh K., Ranade D., Chugh A, & Shastry P. (2016). Suppression of the invasive potential of Glioblastoma cells by mTOR inhibitors involves modulation of NFκB and PKC-α signaling. Scientific Reports, 6, 22455.
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8

Immunoblotting of Signaling Pathways

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Immunoblotting was performed using the following antibodies: α - β -catenin (BD Transduction Labs); α -p4EBP, α -4EBP, α -pS6, α -S6, c-Myc, CREB, Snail (Cell Signaling); α -actin (ImmunO); rabbit α -GSK-3 (Santa Cruz). Reagents used include Torin1, PP 242, BIO, CHIR 99021 (Tocris, >98% purity); BI 6727, GSK461364 (Selleck, >97% purity); LiCl, Rapamycin, Everolimus (Sigma, >95% purity); DMSO was used as vehicle in all experiments unless otherwise stated.
Thorne C.A., Wichaidit C., Coster A.D., Posner B.A., Wu L.F, & Altschuler S.J. (2014). GSK-3 modulates cellular responses to a broad spectrum of kinase inhibitors. Nature chemical biology, 11(1), 58-63.
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9

Investigating Lysosomal Calcium Regulation

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Reagents and chemicals used were LLOMe (L7393; Sigma-Aldrich), GPN (SC-252858; Scbt), BafA1 (1334; Tocris), PP242 (4257; Tocris), Monensin (M5273; Sigma-Aldrich), human serum (H2918; Sigma-Aldrich), Zymosan (Z4250; Sigma-Aldrich), murine IFNγ (315-05; Peprotech), DAPI (D9542; Sigma-Aldrich), IN-1 (17392; Caymen Chemical), MLi2 (5756; Tocris), and saliphenylhalamide (SaliP), and TRPML1 agonist C8 and ATG7 inhibitor ATG7-IN-3 were kindly provided by Casma Therapeutics. BAPTA-AM (B1205; Thermo Fisher Scientific), nocodazole (M1404; Sigma-Aldrich), GFP-Trap (gtma-20), and control magnetic agarose beads (bmab-20) were obtained from Chromotek.
Cross J., Durgan J., McEwan D.G., Tayler M., Ryan K.M, & Florey O. (2023). Lysosome damage triggers direct ATG8 conjugation and ATG2 engagement via non-canonical autophagy. The Journal of Cell Biology, 222(12), e202303078.
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10

Breast Cancer Cell Line Maintenance Protocol

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All cell lines were obtained from the American Type Culture Collection (ATCC). MCF-7 and MDA-MB-468 (ATCC catalog number: HTB-22 and HTB-132 respectively) breast cancer cell lines were maintained in a 1:1 mixture of DME: F12 medium supplemented with 4 mM glutamine, 100 units/mL each of penicillin and streptomycin, and 10% heat-inactivated fetal bovine serum (FBS) and incubated at 37°C in 5% CO2. The MDA-MB-468 inducible expression cells were maintained in the same medium with addition of 50 µg/ml hygromycin and 0.2 µg/mL Puromycin. The HEK-293 cells (ATCC catalog number: CRL-1573) were maintained in DMEM medium with glutamine, antibiotics and 10% FBS. The cell lines resulted negative for mycoplasma contamination. AZD8055 was obtained from AstraZeneca Pharmaceuticals, rapamycin was purchased from EMD Bioscience. RAD001, KU006, WY354, PP242, MLN0128 were purchased from Tocris. Doxycycline was purchased from Sigma Aldrich. Puromycin and hygromycin stock solution were purchased from Invitrogen. Drugs were dissolved in DMSO to yield 10 mM stock and stored at −20°C.
Rodrik-Outmezguine V.S., Okaniwa M., Yao Z., Novotny C.J., McWhirter C., Banaji A., Won H., Wong W., Berger M., de Stanchina E., Barratt D.G., Cosulich S., Klinowska T., Rosen N, & Shokat K.M. (2016). Overcoming mTOR Resistance Mutations with a New Generation mTOR Inhibitor. Nature, 534(7606), 272-276.
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