Inhibitors were prepared as 10 mM stock solutions in DMSO and stored at -20 °C. The GV stage oocytes were isolated from ovaries of ~PD42 female mice and cultured in M2 medium under paraffin oil at 37 °C, 5% CO2 in air. For in vitro treatment of UNC0638, fully-grown GV oocytes were collected and transferred into M2 medium containing UNC0638 (10 μM, Sigma, U4885). The GV oocytes cultured in M2 medium containing equivalent DMSO were used as control. For in vitro treatment of reversine, fully-grown GV oocytes were collected and transferred into M2 medium. 500 nM reversine was added at GVBD.
Unc0638
Manufactured by Merck Group
Sourced in United States, Germany
UNC0638 is a small molecule inhibitor developed by Merck Group. It is designed to target a specific biological pathway or mechanism, but a detailed description of its core function is not available at this time.
Automatically generated - may contain errors
Lab products found in correlation
Bix 01294, by Merck Group (9 mentions)
Apicidin, by Merck Group (3 mentions)
Gsk lsd1, by Merck Group (3 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (2 mentions)
Sirtinol, by Merck Group (2 mentions)
Jib 04, by Merck Group (2 mentions)
Mithramycin a, by Merck Group (2 mentions)
Gsk 126, by Thermo Fisher Scientific (2 mentions)
Gsk j4, by Abcam (2 mentions)
Ly294002, by Merck Group (2 mentions)
Gsk343, by Merck Group (2 mentions)
Alexa fluor 594 goat anti rabbit igg h l antibody, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 goat anti mouse igg h l antibody, by Thermo Fisher Scientific (2 mentions)
Histone h3, by Cell Signaling Technology (2 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Gsk126, by Chemietek (2 mentions)
S2101, by Merck Group (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Dulbecco s modified eagle s medium, by Thermo Fisher Scientific (1 mentions)
A 366, by Merck Group (1 mentions)
27 protocols using unc0638
1
Oocyte Maturation Inhibition
2
NIH3T3 Cell Culture and Compound Treatment
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NIH3T3 cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco, USA) supplemented with 10% fetal bovine serum (Gibco, USA) and 1% penicillin/streptomycin (Gibco, USA). Cells were incubated at 37 °C in a humidified 5% CO2 incubator. Cells were treated with 1 μM UNC0638 (Sigma, USA) or 2 μM apicidin (Sigma, USA) for 96 or 48 h, respectively, and compound-containing media was refreshed every 24 h.
3
Histone Methylation Inhibitor Evaluation
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The HMT inhibitors used were Bix01294 (Sigma‐Aldrich, #B9311), UNC0638 (Sigma‐Aldrich, #U4885), and A‐366 (Sigma‐Aldrich, #SML4110). The HMT inhibitors were dissolved in DMSO and tested in the 1–10 micromolar range to identify concentrations that reduce Histone H3K9me3 levels with minimal toxicity (Bix01294, 2 µM, 24 hr; UNC0638, 3 µM, 24 hr; A‐366, 3 µM, 72 hr). The Zmpste24 protease inhibitor lopinavir (LPV; Cayman Chemicals, #13854) was dissolved in DMSO and used as described (20 µM, 72 hr) to inhibit proteolytic processing of prelamin A. Human fibroblasts were exposed to ionizing radiation (5 Gy), returned to the incubator for 30 min, and subsequently analyzed by IF microscopy. The siRNA to the Ran import factor NTF2 (Santa Cruz, #sc‐36105) and a control siRNA (Fisher, #AM4635) were introduced into normal human fibroblasts (80% confluence) at a concentration of 10 µM using Lipofectamine RNAiMAX (Invitrogen). The cells were analyzed ~96 hr post‐transfection.
4
Immune cell stimulation protocol
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RPMI 1640 (Dutch modified; Gibco, Life Technologies, Waltham, MA, USA) was used as culture medium supplemented with 5 μg mL−1 gentamicin (Centraform, Etten‐Leur, the Netherlands), 2 mm l ‐glutamin (Gibco) and 1 mm pyruvate (Gibco). Cells were stimulated with synthetic Pam3SK4 (Pam3Cys; EMC Microcollections, Tübingen, Germany), Escherichia coli LPS (serotype 055:B5, Sigma‐Aldrich, St Louis, MO, USA), β‐1,3‐(d )‐glucan (kindly provided by Professor David Williams of East Tennessee State University, USA), BCG vaccine (InterVax, Canada, or for bladder cancer patients, BCG‐Medac, Medac, Wedel, Germany) and oxLDL, which was isolated from pooled human serum by ultracentrifugation and oxidation by incubating with 20 µmol CuSO4 L−1 for 15 h at 37°C followed by dialysis, as previously described.38 G9a inhibitors BIX‐01294 (1 µM, Sigma) and UNC0638 (1 µM; Sigma) were used. N‐acetyl cysteine (NAC) was used in a concentration of 1 mM (Sigma).
5
Pharmacological Inhibition of Autophagy
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G9a enzymatic inhibitors, BIX-01294 and UNC0638, autophagy-inhibitors, 3-methyadenine (3-MA) and chloroquine (CQ) were purchased from Sigma–Aldrich, St. Louis, MO, USA. MAPK/ERK kinase inhibitor, U0126, was purchased from Cell Signaling Technology, Danvers, MA, USA.
6
Small Molecule Inhibition Screening
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For all small molecule inhibition studies, the drug was added on the day of encapsulation and replaced with each subsequent media change. The inhibitors used were GSK 126 (100 nM, Fisher Scientific), UNC 0638 (250 nM, Sigma), JIB-04 (1 μM, Sigma), GSK-J4 (10 μM, Abcam), GSK-LSD1 (100 nM, Sigma), Sirtinol (50 μM, Sigma), apicidin (1 μM, Sigma), mithramycin A (50 nM, Sigma), LY294002 (20 μM, Sigma) and SAHA (1 μM, Sigma). All drugs were dissolved in DMSO and diluted in basal medium before adding to the culture medium. DMSO alone was added to the culture medium as a vehicle control.
7
Modulating HIV-1 Reactivation in Microglia
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TNF-α (Invitrogen, Cat. #PHC3015) was used to induce HIV-1 reactivation in microglial cells. Nor1 and Nurr1 agonists 6-mercaptopurine (6-MP) (Millipore-Sigma, Cat#38171) and amodiaquine (AQ) (Millipore-Sigma, Cat#SMB00947) were used to activate the Nerve Growth Factor IB-like nuclear receptors. GSK343 (Sigma Aldrich, Cat# SML0766), UNC0638 (Sigma Aldrich, Cat#U4885), and suberoylanilide hydroxamic acid (SAHA, Millipore-Sigma, Cat#SML0061) were used to examine the effects of EZH2, H9a, and HDAC1/2 on HIV silencing respectively.
8
TBI Treatment with UNC0638 in Mice
9
Chromatin-Modifying Enzyme Inhibitor Protocol
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UNC0638 and BIX01294 were purchased from Sigma (St. Louis, MO, USA), BI2536 was purchased from Selleck (Shanghai, China), BRD9539 and A-366 were purchased from MedChem Express (Shanghai, China). RIPA were purchased from Beyotime (Nantong, China). The study included the following primary antibodies: anti-G9a antibody (Cell Signaling Technology, Danvers, MA, USA), anti-Plk1 antibody (Cell Signaling Technology), rabbit anti-p53 antibody (Abcam, Cambridge, MA, USA), mouse anti-p53 antibody (Santa Cruz Biotechnology, Dallas, USA), anti-pan-methyl lysine antibody (Abcam), anti-histone H3 antibody (Santa Cruz Biotechnology), anti-histone H3 dimethyl (K9) antibody (Cell Signaling Technology), anti-β-actin antibody (Cell Signaling Technology), anti-GAPDH antibody (Cell Signaling Technology), and anti-GFP antibody (Santa Cruz Biotechnology). Horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit secondary antibodies were purchased from KangChen Bio-Tech (Shanghai, China), and HRP-conjugated donkey anti-goat IgG was purchased from Santa Cruz Biotechnology. Protein A/G agarose beads were purchased from Santa Cruz Biotechnology, and protease inhibitor cocktail was purchased from Roche Applied Science (Basel, Switzerland).
10
Quantification of GFP Expression in HIV-Latent Cells
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Quantification of GFP expression was performed by flow cytometry analysis using a LSR Fortessa instrument, the FACSDiva software (BD, NJ) for data collection, and the WinList 3D software for data analysis. Prior to analysis, CHME-5/HIV and HC69 cells (adherent) were trypsinized, collected, and resuspended in 300 μL of cold PBS, while the suspension cells 2D10 and HA3 were centrifuged, and pellets resuspended in 300 μL of PBS. Drug treatments of HIV-latently infected CHME-5/HIV, HC69, HA3, and 2D10 cells were typical for 16 h, with the following concentrations: 4 μM BIX01294 (Sigma-Aldrich B9311), 3 μM UNC0638 (Sigma-Aldrich U4885), 500 μM phenelzine (Sigma-Aldrich P6777), 100 μM M-30 (Sigma-Aldrich SML0128), 100 μM RN-1 (Tocris 4977), 100 μM GSK-LSD1 (Sigma-Aldrich SML1072), and 2 μM SP-2509 (Cayman Chemical 15487).
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