MNT-1 cells were plated in 6-well plates at a concentration of 2 x 105 cells per well and allowed to adhere overnight. Cells were then incubated with varying concentrations of YM-201636 (Cayman Chemical), Apilimod (US Biological), or vehicle control (DMSO) in normal MNT-1 media. At the conclusion of the treatment, cells were lysed with RIPA buffer for downstream analysis. In treatment assays that exceeded 48 hours, media containing drug was refreshed every 48 hours.
Ym201636
Manufactured by Cayman Chemical
Sourced in United States
YM201636 is a chemical compound produced by Cayman Chemical. It is a selective and reversible inhibitor of the mitogen-activated protein kinase (MAPK) signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Apilimod, by US Biological (1 mentions)
Fk866, by Selleck Chemicals (1 mentions)
Dulbecco modified eagle medium (dmem), by Merck Group (1 mentions)
Nad nadh glo assay, by Promega (1 mentions)
Dynasore, by Cayman Chemical (1 mentions)
Coelenterazine h, by Cayman Chemical (1 mentions)
Gw4869, by Cayman Chemical (1 mentions)
Thapsigargin, by Merck Group (1 mentions)
Perphenazine, by Merck Group (1 mentions)
Retro 2, by Merck Group (1 mentions)
Apilimod, by Selleck Chemicals (1 mentions)
Azd8055, by Cayman Chemical (1 mentions)
Secinh3, by Cayman Chemical (1 mentions)
Enzastaurin, by MedChemExpress (1 mentions)
Pac 1, by Selleck Chemicals (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Infinite m200, by Tecan (1 mentions)
Phloretin, by Merck Group (1 mentions)
Xenolight d luciferin k salt, by PerkinElmer (1 mentions)
12 protocols using ym201636
1
Cytotoxicity Screening of YM-201636 and Apilimod
2
NAD+ and NADH Quantification in MEFs
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MEFs (WT and p32KO) were obtained in our laboratory (Yagi et al, 2012 ). All cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM; 1,000 mg/l glucose; Sigma–Aldrich, St. Louis, MO, USA) supplemented with 10% FBS at 37°C in a humidified atmosphere with 5% CO2. MEFs were incubated with the Nampt inhibitor, FK866 (10 nM; Selleck Chemicals Llc, Houston, TX, USA), for 24–48 h in 5% CO2 at 37°C, followed by measurement of total cellular NAD+ and NADH content using the NAD/NADH‐Glo™ Assay (Promega). YM201636 (13576) was purchased from Cayman Chemical. These compounds were solubilized in DMSO (Sigma–Aldrich, D8418), which was used as vehicle control.
3
Lipid Modulator Effects on Cellular Processes
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The compounds used in this study were as follows: pilipin III (C35H58O11 ≥ 85%), Cytochalasin H (C30H39NO5 ≥ 95%), Coelenterazine-h (C26H21N3O2 ≥ 95%), dynasore (C18H14N2O4 ≥ 95%), GW4869 (hydrochloride hydrate, ≥95%), YM-201636 (C25H21N7O3 ≥ 95%), obtained from Cayman Chemical Company (Ann Arbor, Michigan, USA). Thapsigargin (C34H50O12, ≥95%) and sulfatase (EC: 232-772-1) were purchased from Sigma Aldrich (St. Louis, MO, USA). Sulfatides (C42H80NNaO11S, bovine source, ≥98%) were purchased from Matreya LLC (State College,PA, USA). Cytochalasin H, pilipin III, Coelenterazine-h, and dynasore were added 1 h before addition of conditioned PBS and remain present in the medium. Sulfatide was added together with conditioned PBS and kept in the medium. Cytochalasin H, pilipin III, Coelenterazine-h, and dynasore were added 1 h before addition of conditioned PBS and kept in the medium. Sulfatide were added together with conditioned PBS and kept in the medium.
4
Synthesis and Characterization of SH-BC-893
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SH-BC-893 was synthesized by IntelliSyn RD (Montreal, Quebec, Canada) following the methods in (44 (link)) and with advice from S. Hanessian. The following chemicals were purchased: YM201636 (Cayman Chemicals, cat# 13576), apilimod (SelleckChem, cat# S6414), NAV2729 (R&D systems, cat# 5986), SecinH3 (Cayman Chemicals, cat# 10009570), perphenazine (PPZ, Sigma, cat# P6402-1G), UNC10217938A (Medchemexpress, cat# HY-136151), 6BIO (Cayman Chemicals, cat# 13123), AZD8055 (Cayman Chemicals, cat# 16978) and retro-2 (Sigma, cat# SML1085-5MG). Stock solutions were prepared as follows, aliquoted, and stored at –20°C: SH-BC-893 (5 mM in H2O), YM201636 (1.6 mM in DMSO), apilimod (100 μM in DMSO), NAV2729 (12.5 mM in DMSO), SecinH3 (30 mM in DMSO), perphenazine (50 mM in DMSO), UNC10217938A (10 mM in DMSO), 6BIO (15 mM in DMSO), AZD8055 (1 mM in DMSO), and retro-2 (100 mM in DMSO). All chemical structures of compounds are shown in Supplementary Figure S1 . Oligonucleotides used are shown in Supplementary Tables 1, 3 and 4 and were obtained from Ionis Pharmaceuticals.
5
MTT Assay for Cell Viability
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For drug sensitivity evaluation, cells were seeded in a 96-well plate at 4000 cells/well. The next day, cells were incubated at 37 °C with different concentrations of the test compounds. Control cultures were incubated with dimethyl sulfoxide (DMSO). After 48 or 72 h, media were replaced with fresh media supplemented with MTT (Sigma) at a final concentration of 0.5 mg/mL. After 1.5 h, media were removed and formazan crystals were dissolved with 100 μl of DMSO. Absorbance at 570 nM was read in the spectrophotometer (TECAN Infinite M200). The drugs used in this assay were PAC-1 (S2738, Selleckchem), YM201636 (ref. 13576, Cayman Chemical), and enzastaurin (ref. HY-10342, MedChemExpress).
For viability assays after RNA interference, cells were seeded at 4000 cells/well and transfected at the same time as explained in the previous section. After 6 h, media were replaced with fresh new media. MTT assay was performed as explained above each day and viability was compared to T0. All experiments were repeated at least three times with different cell passage number, with three replicates per experiment.
For viability assays after RNA interference, cells were seeded at 4000 cells/well and transfected at the same time as explained in the previous section. After 6 h, media were replaced with fresh new media. MTT assay was performed as explained above each day and viability was compared to T0. All experiments were repeated at least three times with different cell passage number, with three replicates per experiment.
6
Evaluating YM201636 Effects on NSCLC Cell Lines
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To compare the effects of YM201636, we used three NSCLC cell lines (Calu-1, H1299, and HCC827) with different genotypic backgrounds. Calu-1 and H1299 are carcinoma cell lines carrying homozygous TP53 deletion and lack of expression of p53 protein. HCC827 cell line possesses EGFR mutation (exon19del E746-A750), which leads to constitutively activated EGFR.
All cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, USA). Cell culture media and supplements were obtained from Biological Industries (Bio Ind. Kibbutz Beit Haemek, Israel). Calu-1 cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium); HCC827 and H1299 cells were cultured in RPMI-1640 medium in a humidified 37 °C incubator with 5% CO2. Both medias were supplemented with 10% FBS (fetal bovine serum), 1% L-glutamine, and penicillin-streptomycin.
YM201636 was obtained from Cayman Chemical (#13576) and dissolved in DMSO (final concentration 10 mM). For mRNA expression analysis primer, probe and enzymes were purchased from Roche Applied Science (Mannheim, Germany).
All cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, USA). Cell culture media and supplements were obtained from Biological Industries (Bio Ind. Kibbutz Beit Haemek, Israel). Calu-1 cells were cultured in DMEM (Dulbecco’s modified Eagle’s medium); HCC827 and H1299 cells were cultured in RPMI-1640 medium in a humidified 37 °C incubator with 5% CO2. Both medias were supplemented with 10% FBS (fetal bovine serum), 1% L-glutamine, and penicillin-streptomycin.
YM201636 was obtained from Cayman Chemical (#13576) and dissolved in DMSO (final concentration 10 mM). For mRNA expression analysis primer, probe and enzymes were purchased from Roche Applied Science (Mannheim, Germany).
7
Synthesis of Indolyl Chalcone Compounds
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Indolyl chalcone compounds; MOMIPP [9 (link)], MOP IPP [21 (link)], 2a and 2q [22 (link)] were synthesized as described previously. SP600125 and YM201636 were obtained from Cayman Chemical, Ann Arbor, MI. Bafilomycin-A1, n-methylpyrrolidone (NMP), 5-(N-Ethyl-N-isopropyl)amiloride (EIPA), oligomycin, phloretin, cytochalasin B, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) and Solutol-HS15 were from Sigma-Aldrich, St. Louis, MO. XenoLight D-Luciferin-K+ salt was obtained from Perkin Elmer, Waltham, MA.
8
Antibody Validation and Signaling Pathway Analysis
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The following antibodies were used: rabbit anti-p70S6K (Cell Signaling Technologies; #2708), rabbit anti-p-p70S6K (Thr389) (Cell Signaling Technologies; #9205), rabbit anti-Akt (Cell Signaling Technologies; #9272), rabbit anti-p-Akt (Thr308) (Cell Signaling Technologies; #13038), rabbit anti-p-Akt (Ser473) (Cell Signaling Technologies; #4051), rabbit anti-ULK1 (Cell Signaling Technologies; #8054), rabbit anti-p-ULK1 (Ser757) (Cell Signaling Technologies; #6888), rabbit anti-TFEB (Cell Signaling Technologies; #4240), rabbit anti-p-TFEB (Ser211) (Cell Signaling Technologies; #37681), rabbit anti-4E-BP1 (Cell Signaling Technologies; #9644), rabbit anti-p-4E-BP1 (Thr37/46) (Cell Signaling Technologies; #2855), rabbit anti-mTOR (Cell Signaling Technologies; #2983), rabbit anti-Raptor (Cell Signaling Technologies; #2280), rabbit anti-HA (Cell Signaling Technologies; #3724), mouse anti-α-tubulin (Thermo; 236-10501), mouse anti-GAPDH (Thermo; AM4300), mouse anti-DYKDDDDK (WAKO; clone 1E6), mouse anti-LAMP1 (Development Studies Hybridoma Bank; H4A3), mouse anti-PIKfyve (Development Studies Hybridoma Bank; 3C9), rabbit anti-TFE3 (Sigma; HPA023881), mouse anti-PP2A-Cα/β (Santa Cruz; sc-80665), and rabbit anti-PPP2CA (Proteintech; 13482-1-AP). Apilimod was from MedChem Express. Okadaic acid was from Santa Cruz. Torin1, YM201636, VPS34-IN1, and FK-506 were from Cayman Chemical.
9
Modulation of Mitf Signaling Pathways
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hNrg1–1 (25 ng/mL) (Cell Signaling Technology, Catalog # 5218SC); VPS34-IN1 (1 μM) (EMD Millipore, Catalog # 5326280001); YM201636 (2 μM) (Cayman Chemical, Catalog # 13576–1); Canertinib (5 μM) (Selleck Chemicals LLC, Catalog #S1019); MK-2206 (1 μM) (Selleck Chemicals LLC, Catalog #S1078); Torin (5 μM) (APExBIO, Catalog # A8312); Enzastaurin (5 μM) (Selleck Chemicals LLC, Catalog #S1055); Trametinib (5 μM) (Selleck Chemicals LLC, Catalog #S2673); KT5720 (Sigma-Aldrich, Catalog # 420323); Sch772984 (5 μM) (APExBIO, Catalog #B5866); BIO (10 μM) (GSK3, Sigma-Aldrich Inc Cat# B1686); Rapamycin (0.27 μM) (mTorc1, Thermo Scientific Chemicals, Catalog #J67452.XF). Rabbit polyclonal Mitf antibody was raised in house (Animal # 7414, # 7416) against antigens DLVNRIIKQEPVLENCSQE (N-term) and CGTMPESSPAYSIPRKMGSNLEDILMD (C-term), respectively, each independently conjugated to KLH (Thermo Scientific, Cat# 77671). Commercial antibodies: anti-Paxillin (BD Biosciences #610051). Anti-Mitf (Abcam, ab122982), anti-GAPDH (Fitzgerald Cat # 10R-G109a), anti-GFAP (Aviva Cat # OAPC00115), anti-GFAP (Aviva Cat# OAEB01041), anti-S100 (Abcam Cat# ab868), anti-Sox10 (R and D systems, Cat# AF2864). Alexa-conjugated secondary antibodies (Thermo Fisher Scientific).
10
Dose-Dependent PI(3,5)P2 Treatment
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YM201636 (1 mM; #13576, Cayman Chemical) was prepared in DMSO. DMSO at the same dilution was added as the control. PI(3,5)P2 (#10008398, Cayman Chemical) was prepared in PBS buffer (pH 7.2). All experiments were repeated at least three times.
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