MiaPaCa2, MDA-MB231, Panc1, AsPC1, and HEK293T cell lines were obtained from American Type Culture Collection (ATCC) and mycoplasma-free. These cells were cultured in Dulbecco’s minimal essential medium (DMEM) from Nacalai (California, USA) supplemented with 10% v/v FBS and penicillin (100 U/mL)/streptomycin (100 μg/mL) from Hyclone (IL, USA). Antibodies for GAPDH (14C10, #2118), phospho-AKT Ser473 (#9271), phospho-ERK Thr202/Tyr204 (#9101), phospho-S6 Ser235/236 (#2211), YAP (#4912), phospho-YAP Ser127 (#4911), and phospho-LATS1 Ser909 (#9157) were from Cell Signaling Technology (MA, USA). Antibody for TAZ (#HPA007415) was from Sigma-Aldrich (MO, USA). Gemcitabine-HCl (#S1149) was obtained from Selleck Chemicals (TX, USA), while doxorubicin-HCl (#D-4000) from LC Laboratories (MA, USA). PD184352, Triciribine and Rapamycin were obtained from Sigma-Aldrich (MO, USA). The Cell viability was assayed by colorimetric based CellTiter 96® AQueous One Solution Cell Proliferation kit (#G3581, Promega), per manufacture’s protocol.
Pd184352
Manufactured by Merck Group
Sourced in United States
PD184352 is a small-molecule inhibitor that targets the MEK1/2 enzymes. It is a laboratory research tool used to study the role of the MAPK/ERK signaling pathway in cellular processes. The core function of PD184352 is to inhibit the enzymatic activity of MEK1/2, which are key regulatory kinases in the MAPK/ERK cascade.
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Lab products found in correlation
Sp600125, by Merck Group (6 mentions)
Sb203580, by Merck Group (4 mentions)
Wortmannin, by Merck Group (3 mentions)
Hepatocyte growth factor (hgf), by Merck Group (2 mentions)
Dmem 10 fbs, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Mk 2206, by Selleck Chemicals (2 mentions)
Xav939, by Merck Group (2 mentions)
Pictilisib, by Selleck Chemicals (2 mentions)
Sp600125, by Bio-Techne (2 mentions)
Sb203580, by Bio-Techne (2 mentions)
Y 27632, by Merck Group (2 mentions)
Isoproterenol, by Merck Group (2 mentions)
Cid755673, by Bio-Techne (2 mentions)
Mia paca 2, by American Type Culture Collection (1 mentions)
Hek293t, by American Type Culture Collection (1 mentions)
Mda mb 231, by American Type Culture Collection (1 mentions)
Phospho s6 ser235 236, by Cell Signaling Technology (1 mentions)
Phospho erk thr202 tyr204, by Cell Signaling Technology (1 mentions)
Panc 1, by American Type Culture Collection (1 mentions)
31 protocols using pd184352
1
Anticancer Drug Screening in Cell Lines
2
Signaling Pathways Modulate Gap27 Gene Expression
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To determine the role of key signaling pathways in Gap27-induced gene expression, we blocked TGF-β pathway with SB431542 (20 μM; Selleckchem, Houston, TX, USA), MEK1/2 with PD184352 (10 μM; Sigma-Aldrich), p38 with SB203580 (10 μM; Cell Signaling, Danvers, MA, USA), GSK3α/β with SB415286 (30 μM; Biomol, Hamburg, Germany), AP1 with curcumin (30 μM; Sigma-Aldrich), and SP1 with WP631 (5 nM; Sigma-Aldrich) in Gap27-treated cells, respectively. To this end, confluent GFBL cultures were pre-incubated with inhibitors at 37°C for 1 h, and then treated with Gap27 (150 μM) with or without the inhibitors in serum-free growth medium for 24 h. All inhibitors were dissolved in DMSO, and control samples were treated with respective amounts of DMSO only. Total RNA was collected for real-time PCR as described above.
3
Whey Protein Modulation of Osteoblast Genes
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Saos-2 cells were seeded at a density of 1 × 106 cells/well in 100 mm culture dishes. Cells were grown in serum-free medium and incubated with WP 0.5; 1; 2 mg/mL for 24 h. Total RNA from cells were extracted with Trizol reagent (Life Technologies, Cramlington, UK) according to the manufacturer’s instructions. cDNA was synthesized from 1 µg total RNA, using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). mRNA expression of RANKL, OPG, RUNX2, COL1A, and β-ACTIN were quantified by real time-PCR using SYBR® Green dye (SYBR® Green PCR Master Mix, Applied Biosystems, Foster City, CA, USA) (Supplementary Table S2 ). Moreover, Saos-2 cells were treated with a high dose of 2 mg/mL of WP and 40 μM of MEK/ERK1/2 inhibitor PD184352 (#PZ0181, Sigma Aldrich, St. Louis, MO, USA) [28 (link)].
4
MASM Synthesis and Characterization
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MASM [(6aS,10S,11aR,11bR,11cS)-10-methylamino-dodecahydro-3a,7a-diazabenzo (de)anthracene-8-thione] (purity > 99%) was synthesized and characterized as reported earlier [23 (link)]. Chloroquine (Sigma, Germany), N-Acetyl-L-cysteine (NAC, Sigma, Germany) were dissolved in phosphate buffered saline (PBS). LY294002 (Invivogen, Germany), Wortmaninn (Invivogen, Germany), PD184352 (Sigma, Germany), SB230580 (adooq biosciences, USA) were dissolved in dimethyl sulfoxide (DMSO).
5
Inhibition of MAPK and NF-κB Signaling
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The MEK inhibitor PD184352 (Sigma-Aldrich, St. Louis, MO, USA) was used at a final concentration of 10 μM, which led to successful p-ERK inhibition [22 (link)]. The AKT inhibitor MK2206 (Deltaclon S.L., Madrid, Spain) was used at a final concentration of 1 µM [23 (link)]. Parthenolide (Sigma-Aldrich) and Amgen16 (Sigma-Aldrich) were used for NF-ĸB canonical and noncanonical inhibition, respectively, at 10 µM [24 (link),25 (link)]. PC-3 cells were treated with inhibitors for 1 h before stimulation with 100 ng/mL TWEAK (or vehicle) for 24 h. RNA was then extracted for gene expression analysis.
6
Retinoid Effects on Cell Proliferation
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For proliferation studies, overnight serum-starved cells were treated with different concentrations of atRA and ROL (Sigma-Aldrich) in 0.1% FBS up to 6 days. For cell viability, 3-(4,5 dimethylthiazol-2-yl)-2,5diphenyl-tetrazolimbromide assay (MTT; Sigma-Aldrich) was carried out in triplicate ([44 (link)]. In some instances, CRBP-1+ cells were pre-treated with selective EGFR (AG1478; Sigma-Aldrich; 10 μM), phosphatidylinositol 3-kinase/ AKT (Wortmannin; Sigma-Aldrich; 10 μM) and the mitogen-activated protein kinase kinase (MAPKK, PD184352; Sigma-Aldrich; 2μM) inhibitors.
7
Melanoma Cell Line Characterization and Treatment
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Human melanoma lines (A375, mel-537, mel-624, mel-888, SKMEL-5, SKMEL-23, and SKMEL-28), HEK293T, and 3T3 cells (ATCC, Manassas, VA and University of Chicago Comprehensive Cancer Center Core Facilities) were cultured in DMEM/10% FBS (Sigma-Alrich, St. Louis, MO, USA). Morphology, growth curve analysis, and melanoma-marker testing verified melanoma cell identity. All cells were negative for the presence of mycoplasma. For Hepatocyte Growth Factor (HGF) treatment, cells were grown in serum-free media, supplemented with 0.5% bovine serum albumin (BSA) and 30 ng/ml HGF (EMD-Millipore, Temecula, CA) for 7.5–10 minutes prior to cell collection and analysis. For PD184352 (Sigma-Alrich) treatment, 1 μM was added for the length of the experiment.
8
Resveratrol Modulation of Kinase Pathways
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Resveratrol, the inhibitors of kinases (SB202190 for p38 MAPK, SP600125 for JNK, PD184352 for ERK, wortmannin for PI3K) and SIRT1 (EX-527), caspase 3 activity assay kit using fluorogenic caspase 3 substrate (Ac-DEVD-AMC), buffer components and N-acetylcysteine were purchased from Sigma-Aldrich (St. Louis, MO, USA). Non-selective caspase inhibitor (Ac-VAD-CMK) was obtained from AnaSpec (Fermont, CA, USA) and CytoxOne lactate dehydrogenase release kit from Promega (Fitchburg, WI, USA). Cell culture mediums and fetal bovine serum were supplied by GE Healthcare (Little Chalfont, UK) and Life Technologies (Carlsbad, CA, USA), respectively. Test compounds were dissolved in DMSO and used in cell culture medium to provide 0.5% final DMSO concentration. Control cells were treated with the same concentration of DMSO.
Pregnant NMRI mice were supplied by Toxicoop, Gödöllő, Hungary. All animal procedures were approved by the ethics committee of the Semmelweis University (22.1/606/001/2010, February 5, 2010) and were in accordance with the EU Council directives on laboratory animals (86/609/EEC).
Pregnant NMRI mice were supplied by Toxicoop, Gödöllő, Hungary. All animal procedures were approved by the ethics committee of the Semmelweis University (22.1/606/001/2010, February 5, 2010) and were in accordance with the EU Council directives on laboratory animals (86/609/EEC).
9
Chronic myeloid leukemia cell line K-562 inhibition
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Chronic myeloid leukaemia cell line K‐562 was cultured in RPMI‐1640 media (Invitrogen, Thermo Scientific) with 10% FBS (HiMedia) at 37°C in 5% CO2. Imatinib mesylate (Sigma–Aldrich; SML1027), dasatinib (Sigma–Aldrich, SML2589), and bosutinib (Bonitar 100 mg) were used as inhibitors for BCR‐ABL1 protein. PD184352 (Sigma–Aldrich; PZ0181), A‐395 (Adooq Biosciences; A12999), and IPA‐3 (Sigma–Aldrich; I2285) were used as inhibitors for MEK/ERK kinase, PRC2 complex, and PAK1 kinase respectively. Antibodies used were FUBP3 (sc‐398,466; E8), GAPDH (CST‐14C10), Histone H3 (CST‐D1H2), beta‐tubulin (Sigma–Aldrich N6786), p44/42 MAPK (ERK1/2) (CST‐137F5), phospho‐p44/42 MAPK (ERK1/2) (CST‐D13.14.4E), p38 MAPK (CST‐D13E1), phospho‐p38 MAPK (CST‐D3F9), SAPK/JNK (CST‐9252), phospho‐SAPK/JNK (CST‐81E11), SUZ12 (CST‐D39F6), EZH2 (CST‐D2C9), EED (CST‐E4L6E), RBBP4/7 (RBAP48/RBAP46) (CST‐4633), Mouse IgG (CST‐G3A1), Mouse HRP (Sigma–Aldrich A3682) and Rabbit HRP (Sigma–Aldrich AQ132P).
10
Investigating Endoplasmic Reticulum Stress in HepG2 Cells
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Human hepatoma (HepG2) cells (ATCC, Mannasas, VA) were cultured in Dulbecco's modified Eagle medium (DMEM) with 10% fetal bovine serum and maintained at 37°C in 5% CO2. Cells were grown to 80% confluence in 6-well plates and treated with 12 μmol/L tunicamycin, 100 nmol/L thapsigargin (Sigma-Aldrich, St. Louis, MO), 5 mmol/L DL-homocysteine (Sigma-Aldrich), or vehicle (DMSO/saline) in serum-free DMEM for 6 hours. To determine whether the effects of tunicamycin are dependent on c-Jun-N-terminal kinase (JNK) or extracellular signaling-regulated kinase (ERK), HepG2 cells were treated with the JNK inhibitor SP600125 (Sigma-Aldrich) at a concentration of 25 μmol/L or the MAPK/ERK kinase inhibitor PD184352 (Santa Cruz Biotechnology, Dallas, TX) at 1 μmol/L or vehicle (DMSO/saline) as previously described.26 (link) One hour after exposure to SP600125 or PD184352, cells were treated with tunicamycin (12 μmol/L; Sigma-Aldrich) in serum-free DMEM and incubated for an additional 6 hours. Successful inhibition of JNK and ERK activation was confirmed by Western blot analysis as previously described.26 (link)
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