Bone marrow cells were obtained from femurs and tibias of 8-week-old O-ND and O-HFD mice and cultured in complete medium (α-MEM (Dulbecco’s Modified Eagle’s Medium) supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin, and 40-ng/mL macrophage-colony stimulating factor-1 (M-CSF)). Nonadherent cells were collected after 24 h and differentiated into bone marrow-derived macrophages (BMDMs) using 40-ng/mL M-CSF for seven days, yielding 98% F4/80+ cells. For differentiation into osteoclast-like macrophages, BMDMs were stimulated with 100-ng/mL tumor necrosis factor-α (TNF-α; PEPROTECH, Rocky Hill, NJ, USA) and 40-ng/mL M-CSF for 3 days [17 (link)]. For the enhancer of zeste homolog 2 (EZH2) inhibitor treatment or ZA treatment, 10-μM GSK126 (Abcam) or 0.03-μM ZA (Sigma Aldrich) were added to the complete medium during the M-CSF-induced BMDM expansion phase and TNF-α-induced osteoclast differentiation phase. For the classical BMDM polarization assay, BMDMs were stimulated with either 100-U/mL lipopolysaccharide (LPS) (Sigma-Aldrich) or 1-μg/mL elastin-derived peptides (EDP, COSMO BIO, Tokyo, Japan) [21 (link)].
Gsk126
Manufactured by Abcam
Sourced in United States
GSK126 is a highly potent and selective inhibitor of the EZH2 histone methyltransferase enzyme. It is used in research applications to study the role of EZH2 and epigenetic regulation.
Automatically generated - may contain errors
Lab products found in correlation
Epz 6438, by Selleck Chemicals (3 mentions)
Lncap, by American Type Culture Collection (2 mentions)
Intesticult organoid growth medium, by STEMCELL (2 mentions)
Matrigel, by Corning (2 mentions)
Tumor necrosis factor α tnf α, by Thermo Fisher Scientific (1 mentions)
Tnf α, by Merck Group (1 mentions)
Lipopolysaccharide lps, by Merck Group (1 mentions)
Nerve growth factor (ngf), by Thermo Fisher Scientific (1 mentions)
Dibutyryl adenosine 3 5 monophosphate camp, by Merck Group (1 mentions)
Brain derived neurotrophic factor (bdnf), by Thermo Fisher Scientific (1 mentions)
Ascorbic acid, by Merck Group (1 mentions)
Mg132, by Merck Group (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Xav939, by Abcam (1 mentions)
Pd0325901, by Merck Group (1 mentions)
Chir99021, by Abcam (1 mentions)
Sfrp1, by R&D Systems (1 mentions)
Ag490, by InvivoGen (1 mentions)
Activin a, by R&D Systems (1 mentions)
Basic fibroblast growth factor (bfgf), by Thermo Fisher Scientific (1 mentions)
11 protocols using gsk126
1
Osteoclast Differentiation from Bone Marrow Macrophages
2
Differentiation of hENCCs into Neurons using Ezh2 Inhibition
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hENCCs were derived from human pluripotent stem cell (hPSCs) and then directed to neuronal lineage for generation of human enteric NPs, as described previously (29 (link), 36 (link), 43 (link)). To examine the effect of Ezh2 on neuronal differentiation of hENCCs, hENCCs were cultured in differentiated medium [N2 medium containing brain-derived neurotropic factor (BDNF) (10 ng/ml; PeproTech), GDNF (10 ng/ml; PeproTech), NT-3 (10 ng/ml; PeproTech), nerve growth factor (NGF) (10 ng/ml; PeproTech), 1 μM dibutyryl adenosine 3′,5′-monophosphate (cAMP) (Sigma-Aldrich), and 200 μM ascorbic acid (Sigma-Aldrich)] in the absence or presence of Ezh2 inhibitor (GSK126, 2.5 μM, Abcam or EPZ-6438, 5 μM, AbMole) for 5 days. The differentiation capacity of hENCCs was monitored on the basis of the expressions of TUJ1/HU, as revealed by immunocytochemistry.
3
Differentiation of Cell Lines
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C2C12 myoblasts, HEK293T cells and BJ fibroblasts (obtained from the ATCC and tested for mycoplasma contamination) were propagated in GM (DMEM supplemented with 10% FBS). After 2-3 days cells were induced to differentiate in DM (DMEM supplemented with 2% HS). Where indicated, cells were treated with SB202190 (Calbiochem, 5 μM), MG132 (Sigma, 10 μM), cycloheximide (CHX, Sigma, 20 mg ml−1), GSK-126 (BioVision, 4 μM) and dZNep (BioVision, 5 μM).
4
Prostate Cancer Cell Lines and Epigenetic Modifiers
5
Modulation of Pluripotent Stem Cell Fate
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ESCs were treated with the following reagents in the described experimental procedures: 100 ng ml−1 Dkk1 (R&D, cat. 5897-DK); 100 ng ml−1 Sfrp1 (R&D, cat. 1384-SF); 100 ng ml−1 Wnt-3a (R&D, cat. 5036-WN); 12 ng ml−1 bFGF (Invitrogen, cat. 13256029); 20 ng ml−1 Activin-A (R&D, cat. 338-AC); 3 μM CHIR99021 (AbCam, cat. ab120890); 1 μM PD 0325901 (Sigma, cat. N°PZ0162); 1 μM EPZ005687 (BioVision, cat. 2364); 2 μM GSK126 (BioVision, cat. 2282); 5 μM AG490 (Invivogen, cat. N°tlrl-ag4); 1 μM Jak1 (Millipore, cat. N°420099); 3 μM XAV939 (AbCam, cat. N°ab120897).
6
EZH2 Inhibitor Treatment in C4-2 Cells
7
Prostate Cancer Cell Lines and Epigenetic Modifiers
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Human embryonic kidney cell line 293T and PCa cell lines LNCaP and
22RV1 were obtained from American Type Culture Collection (ATCC) and
C4–2B cells were a provided by Dr. Arul Chinnaiyan (University of
Michigan, Ann Arbor). 293T cells was cultured in DMEM with 10% fetal bovine
serum (FBS) and 1× Penicillin Streptomycin and PCa cells were
cultured in RPMI1640 with 10% fetal bovine serum (FBS) and 1×
Penicillin Streptomycin solution. LAPC4 cells were provided by Dr. C Shad
Thaxton (Northwestern University) and cultured in IMEM with 10% FBS and 1nM
fresh R1881. All cell lines were authenticated (Genetica DNA Laboratories)
and free of mycoplasma. GSK126 was purchased from BioVision (2282–5),
Enz (S1250) and EPZ6438 (S7128) were purchased from Selleck Chemicals.
22RV1 were obtained from American Type Culture Collection (ATCC) and
C4–2B cells were a provided by Dr. Arul Chinnaiyan (University of
Michigan, Ann Arbor). 293T cells was cultured in DMEM with 10% fetal bovine
serum (FBS) and 1× Penicillin Streptomycin and PCa cells were
cultured in RPMI1640 with 10% fetal bovine serum (FBS) and 1×
Penicillin Streptomycin solution. LAPC4 cells were provided by Dr. C Shad
Thaxton (Northwestern University) and cultured in IMEM with 10% FBS and 1nM
fresh R1881. All cell lines were authenticated (Genetica DNA Laboratories)
and free of mycoplasma. GSK126 was purchased from BioVision (2282–5),
Enz (S1250) and EPZ6438 (S7128) were purchased from Selleck Chemicals.
8
OVCAR3 Cell Culture Protocol
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OVCAR3, a representative HGSOC cell line, was maintained at 37°C and 5% CO2 as described previously [6 (link), 9 (link)]. This cell line was authenticated by ATCC in 2018. OVCAR3 cells were cultured in DMEM 1X (Gibco, #11995065) containing 10% FBS (R&D Systems, #S11150) without antibiotics. OVCAR3 cells used in the study were passaged for less than 15 passages. 50 mM stock solution of RAC inhibitor (NSC23766, Sigma #553502) was made in DMSO. 5 mM stock solution of EZH2 inhibitor (GSK126, Biovision #2282) was made in DMSO. For all the experiments using these inhibitors, an equivalent amount of DMSO or RAC1i or EZH2i or combination of both was added to cells and incubated for 48 hrs at 37°C and 5% CO2.
9
Endometriotic Cell Line Proteomic Analysis
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The endometriotic epithelial cell line 11Z2 was kindly provided by Dr. Jung-Hye Choi and was cultured in RPMI-1640 medium (Gibco Laboratories, Grand Island, NY, USA) supplemented with 5% fetal bovine serum (FBS) (Gibco), 100 IU/mL penicillin G, 100 μg/mL streptomycin and 2.5 μg/mL Amphotericin B (Hyclone, Utah, USA). Total cellular proteins were extracted from 11Z cells at 48 h, after the treatment with dimethyl sulfoxide (DMSO) or 3-Deazaneplanocin A (DZNep) (1 or 2.5 μM, Sigma, MO, USA), or the treatment with DMSO or GSK126 (0.5 or 1 μM, Biovision, Mountain View, CA, USA), as previously reported35 (link), 54 (link).
10
Intestinal Organoid Culture with EZH2 Inhibition
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Intestinal organoids were cultured according to the Sugimoto and Sato protocol (Sugimoto and Sato, 2017). The terminal ileum was harvested from C57BL/6J mice at P4, P7, and P10, and the tissues were sliced into segments. The crypts were isolated by 2.5 mM ethylenediaminetetraacetic acid (EDTA) in PBS for 30 min at 4 °C and pelleted by centrifugation at 400 × g for 3 min at 4 °C. The crypts were then resuspended in Matrigel (Corning) and transferred to 48-well plates. After polymerization, mouse IntestiCult organoid growth medium (Stem Cell Technologies) supplemented with penicillin-streptomycin (100 U/mL) was added to each well and incubated at 37 °C. 3 days after crypt isolation, 0.2 µM 3-Deazaneplanocin A hydrochloride (DZNep, Abcam) or 20 µM GSK126 (Bio Vision) was added to the medium to inhibit the activity of histone methyltransferase EZH2, which mediates H3K27 methylation. After 3 days of incubation with the EZH2 inhibitor, the organoids were observed under a light microscope.
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