C2C12 MBs were cultured until ~90% confluence and then induced to differentiate in medium treated with vehicle (VEH; DM supplemented with DMSO; Invitrogen Life Technologies) or treated with TC-E 5003 (TC-E; DM supplemented with (volume; final concentration) 2.1 µl; 0.1 µM TC-E; Tocris, Bristol, United Kingdom), a highly selective and specific antagonist of PRMT1 [23 (link)]. Four experimental timepoints were employed to examine skeletal muscle differentiation, including D1, D3, D5, and D7, after which cells were washed three times with PBS and prepared for analyses.
Tc e 5003
Manufactured by Bio-Techne
Sourced in United Kingdom
The TC-E 5003 is a laboratory instrument designed for the incubation and temperature control of cell cultures. It features a temperature range of 20°C to 50°C and a temperature accuracy of ±0.1°C. The device is equipped with a digital display for temperature monitoring.
Automatically generated - may contain errors
Lab products found in correlation
Hek293t cell, by American Type Culture Collection (2 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (1 mentions)
Flag tag (flag), by Thermo Fisher Scientific (1 mentions)
Phox2b, by Abcam (1 mentions)
Furamidine, by Bio-Techne (1 mentions)
Prmt5, by Merck Group (1 mentions)
Human pancreatic adenocarcinoma cell lines, by American Type Culture Collection (1 mentions)
C jun n terminal kinase jnk, by Santa Cruz Biotechnology (1 mentions)
3 4 5 dimethylthiazol 2 yl 2 5 diphenyltetrazolium bromide mtt, by Avantor (1 mentions)
Polyethylenimine (pei), by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Puromycin, by Merck Group (1 mentions)
Raw264.7 cell, by American Type Culture Collection (1 mentions)
4 protocols using tc e 5003
1
Skeletal Muscle Differentiation Assay
2
Evaluation of PRMT Inhibitors in Neuroblastoma
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Furamidine, pentamidine, hexamidine, and TC-E5003 were purchased from Tocris. Decamidine and SKLB639 were described previously15 (link),17 (link). Antibodies used in this study include PRMT13 (link), PRMT5 (Millipore), aDMA (Cell Signaling), sDMA (Millipore), MYCN3 (link), TH (Millipore), PHOX2B (Abcam), PARP (Cell Signaling), ATF5 (Abcam), Flag (Thermo Fisher Scientific), and β-actin3 (link).
3
Human Pancreatic Adenocarcinoma Cell Lines
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If not otherwise specified, all human pancreatic adenocarcinoma cell lines, HPNE cells, and HEK293T cells were purchased from the American Type Culture Collection. PDA cell lines MDA28 and MDA48 were gifts from Dr. Paul J. Chiao (The University of Texas MD Anderson Cancer Center). Generation of the FG human pancreatic adenocarcinoma cell line was described previously (1) . All cell lines were maintained in 5% CO2 at 37°C as an adherent monolayer in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum, sodium pyruvate, nonessential amino acids, L-glutamine, penicillin/streptomycin, and a vitamin solution (Flow Laboratories). The American Type Culture Collection performs characterization and authentication of the cell lines it provides using short tandem repeat profiling, and the cell lines they provided were passaged in our laboratory for fewer than 6 months after reception. S3. PRMT1 specific inhibitors DB75 and TC-E5003 were purchased from Tocris Bioscience. Etoposide and H2O2 were purchased from Sigma-Aldrich Corporate. Gemcitabine hydrochloride was purchased from United States Pharmacopeia (USP).
4
Macrophage and Kidney Cell Characterization
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TC-E 5003 was purchased from Tocris Bioscience (Bristol, UK). RAW264.7 cells, concatenation of Bagg and Albino (BALB/c)-derived murine macrophage cell line (No. TIB-71), and HEK293T cells and a human embryonic kidney cell line (No. CRL-3216) were acquired from American Type Culture Collection (ATCC) (Rockville, MD, USA). Lipopolysaccharide (LPS, Escherichia coli 0111:B4), l -NAME (NG-Nitro-l -arginine Methyl Ester), puromycin and polyethylenimine (PEI) were obtained from Sigma (St. Louis, MO, USA). 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Amresco (Solon, OH, USA). Fetal bovine serum (FBS) was purchased from Biotechnics Research (Lake Forest, CA, USA), and Roswell Park Memorial Institute 1640 (RPMI1640) and Dulbecco’s Modified Eagle Medium (DMEM) were obtained from HyClone (Grand Island, NY, USA). Antibodies against the total or phosphorylated forms of c-Jun, p65, p50, IκBα, Src, Syk, p38, extracellular signal-regulated kinase (ERK), Lamin A/C, and β-actin were purchased from Cell Signaling (Beverly, MA, USA). The antibodies against the total and phosphorylated forms of c-Fos, c-Jun N-terminal kinase (JNK) and HA were acquired from Santa Cruz Biotechnology Inc. (Dallas, TX, USA).
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