Chaetocin

Manufactured by Selleck Chemicals

Sourced in United States, China

Chaetocin is a fungal metabolite that functions as a histone methyltransferase inhibitor. It can be used as a laboratory research tool to explore epigenetic mechanisms.

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Lab products found in correlation

8 protocols using chaetocin

Isolation and Culture of Mouse MSCs

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MSCs were isolated from the femur and tibia bone marrow of adult c57bl/6 mice as described previously. [14 (link)] Cells were cultured in α-minimum essential medium (α-MEM; Thermo Fisher Scientific, China) supplemented with 10% fetal bovine serum (FBS; Gibco, China) and 100 μg/ml penicillin-streptomycin (Gibco, China) in a humidified atmosphere containing 5% CO₂ at 37°C. For Chaetocin experiment, 5 μM Chaetocin (Selleck, China) was used. DMSO was used as control.

Liang Y., Liu X., Zhou R., Song D., Jiang Y.Z, & Xue W. (2021). Chaetocin Promotes Osteogenic Differentiation via Modulating Wnt/Beta-Catenin Signaling in Mesenchymal Stem Cells. Stem Cells International, 2021, 8888416.

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High-Throughput Screening of PDX Tumor Cells

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Cells dissociated from PDX tumors and depleted of mouse stromal cells were used for high throughput screening. For the primary screen 5093 compounds were dispensed with Janus 384 pintool (Perkin Elmer) into 384 well plates. Cells were plated into the 384 well plates containing screening compounds at 2000 cells/well in Kubota’s medium (PheonixSongs biological) supplemented with 2% p/s and incubated at 37C for 72 hours. Compounds were screened at a final concentration of 1μM. Each plate contained negative control wells treated with DMSO and positive control wells treated with 20 μM chaetocin (Selleckchem #8068). Plates with a Z'-factor <0.5 were disregarded or repeated. The screen was performed in triplicate for FLC1 and FLC5. After 72 hours of incubation Cell titer glo reagent (Promega) was added using Thermo Multidrop Combi (Thermo Scientific) according to the manufacturer’s instructions, and the plates were read for luminescence (BioTek Synergy Neo). The normalized percent cell survival was calculated by

(%)_{survival} = [100] - [(I_{positive} - I_{compound}) ∕ (I_{positive} - I_{negative})^{*} 100] .

Lalazar G., Requena D., Ramos-Espiritu L., Ng D., Bhola P.D., de Jong Y.P., Wang R., Narayan N.J., Shebl B., Levin S., Michalidis E., Kabbani M., Vercauteren K.O., Hurley A.M., Farber B.A., Hammond W.J., Saltsman JA I.I.I., Weinberg E.M., Glickman J.F., Lyons B.A., Ellison J., Schadde E., Hertl M., Leiting J.L., Truty M.J., Smoot R.L., Tierney F., Kato T., Wendel H.G., LaQuaglia M.P., Rice C.M., Letai A., Coffino P., Torbenson M.S., Ortiz M.V, & Simon S.M. (2021). Identification of Novel Therapeutic Targets for Fibrolamellar Carcinoma Using Patient Derived Xenografts and Direct from Patient Screening. Cancer discovery, 11(10), 2544-2563.

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High-throughput Screening of PDX and PHH Cells

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Cells dissociated from FLC PDX tumors were depleted of mouse stromal cells and used for high-throughput screening. Cells were plated into either 384-well plates (Grenier Bio One) or a 96-well plate containing screening compounds at 2,000 cells per well (384-well format) or 20,000 cells per well (96-well format) in Kubota’s medium (Phoenix Songs Biologicals) supplemented with 2% penicillin/streptomycin. Each plate contained negative control wells with DMSO (I_Negative) and positive control wells (I_Positive) treated with 20 μM chaetocin (Selleckchem, S8068). Cells plated with the compounds (I_Compound) were incubated for 48–72 hours at 37°C. After incubation, CellTiter-Glo reagent (Promega) was added at a 1:1 ratio to the total volume with cells and compounds. The plates were read for luminescence (BioTek Synergy Neo). The normalized percent cell survival was calculated as:Dose-response curves were generated with GraphPad PRISM (version 9). Similarly, the compounds were screened against PHH) grown in humanized mice as described previously (14 (link)). PHH cells were plated at 50,000 cells per well in W10 media (William’s E media supplemented with ITS [BD Biosciences], penicillin/streptomycin/ciprofloxacin). PHH cells were incubated and assayed for viability as described for FLC PDX.

Shebl B., Ng D., Lalazar G., Rosemore C., Finkelstein T.M., Migler R.D., Zheng G., Zhang P., Jiang C.S., Qureshi A., Vaughan R., Yarchoan M., de Jong Y.P., Rice C.M., Coffino P., Ortiz M.V., Zhou D, & Simon S.M. (2022). Targeting BCL-XL in fibrolamellar hepatocellular carcinoma. JCI Insight, 7(17), e161820.

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Chromatin Regulation Assay in Drosophila

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Sucrose (#S0389), NaCl (#S3014), CaCl₂ (#C5670), MgCl₂ (#M4880), KCl (#P9541), NaH₂PO₄ (#S3139), NaHCO₃ (#S5761), and HEPES (#54457) were from Sigma-Aldrich. Hexanoic acid (Macklin, #H810882) for PER assay was purchased from Macklin. EED226 (5 μM, Selleck, #S8496), Chaetocin (5 μM, Selleck #S8068), and A-395 (10 μM, Sigma-Aldrich, #SML1923) were added to the standard medium. Flies were kept on these foods for 5 days before the assay (change fresh medium every 2 days).
The following antibodies were used: The antibodies against Histone H3 (di methyl K9) (#ab1220), acetyl histone-h3-k27 (#ab4729), Histone H3 (trimethyl K27) (#ab6002, #PTM-5002, #PTM-647RM), Histone H3 (trimethyl K9) (#ab8898), and Histone H3 antibody (#ab1791) were purchased from Abcam and Jingjie PTM BioLab. The secondary antibodies against rabbit (Alexa Fluor 633, #a21071, HRP-linked Antibody, #111-035-003) and mouse (Alexa Fluor 488, #a11001) were purchased from Jackson ImmunoResearch and Invitrogen. Fluoroshield with DAPI (#F6057) was purchased from Sigma-Aldrich.

Yang J., Tang R., Chen S., Chen Y., Yuan K., Huang R, & Wang L. (2023). Exposure to high-sugar diet induces transgenerational changes in sweet sensitivity and feeding behavior via H3K27me3 reprogramming. eLife, 12, e85365.

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Chromatin Regulation Assay in Drosophila

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Chaetocin-Induced Transcriptional Profiling

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Two hundred thousand cells were seeded on a T25 flask in SFDM. Twenty-four hours later the media was supplemented with a sublethal concentration of Chaetocin (10 nM; Selleckchem, Houston, Texas, USA) and incubated for 72 h. Control samples were treated with DMSO only. After that, RNA was extracted using RNeasy mini kit (Qiagen). RNA libraries were prepared (Illumina NextSeq 500 High output kit v2) and run on the Illumina NextSeq for 75 bp paired end reads. Expression matrix were obtained using Rsubread R package⁵¹. Differential expression analysis was performed with the Limma R package. In addition, principal component analysis (PCA) was computed on the genes with a log2 fold change ≥ 1 and a false discovery rate (FDR) < 0.05. The distance between control and treated PDPCC was computed accounting the coordinates from the dimension with the higher explained variance.

Fraunhoffer N.A., Abuelafia A.M., Bigonnet M., Gayet O., Roques J., Nicolle R., Lomberk G., Urrutia R., Dusetti N, & Iovanna J. (2022). Multi-omics data integration and modeling unravels new mechanisms for pancreatic cancer and improves prognostic prediction. NPJ Precision Oncology, 6, 57.

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Histone Mutant Expression and Epigenetic Enzyme Purification

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Doxycycline (Sigma; 10,000 × stock solution; 1 mg/ml) was resuspended in 70% ethanol. Biotin (BioShop and Sigma) was prepared as a 1000 × stock solution (50 mM) by dissolving 250 mg in 2 ml 30% v/v NH₄OH on ice and neutralized by slowly adding 18 ml HCl (1 N), before sterile filtration through a 0.22 µm filter and storage at 4 °C. Chaetocin (Selleck) and OTS186935 (MedChemExpress) were dissolved in DMSO, aliquoted, and stored at − 80 °C.
H3.3 (H3-3a) or H3.1 (H3C2) cDNA that was WT or had K27M or G34R mutations was amplified from existing cDNA stocks and cloned between the XbaI/BamHI sites of a pCDH-CMV-MCS-EF1α-Hygro (SystemBioscience) previously modified to encode a FLAG/HA tag between the BamHI/NotI sites. K9M mutations were introduced with the Q5 site-directed mutagenesis kit (NEB). pcDNA5-FLAG-BirA* plasmids to create Flp-In T-REx HEK293 cells were generated as previously described [34 (link)]. Bacterial histone expression vectors were created by cloning cDNA into pET3a [17 (link)]. EHMT2 cDNA was amplified from pLenti6-MK1-EHMT2-V5 (Addgene #31113) and cloned between the KpnI and EcoRI sites of pFastBac (Invitrogen) to create pFastBac_EHMT2. SUV39H2 cDNA was amplified from a HEK293T cDNA library and inserted by Gibson assembly into pDest_pACE1 to create pACE1_SUV39H2. All plasmids were sequenced before use. shRNA targeting H3K9 methylases were from Sigma.

Siddaway R., Canty L., Pajovic S., Milos S., Coyaud E., Sbergio S.G., Vadivel Anguraj A.K., Lubanszky E., Yun H.Y., Portante A., Carette S., Zhang C., Moran M.F., Raught B., Campos E.I, & Hawkins C. (2022). Oncohistone interactome profiling uncovers contrasting oncogenic mechanisms and identifies potential therapeutic targets in high grade glioma. Acta Neuropathologica, 144(5), 1027-1048.

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Chaetocin Effects on Hepatocytes

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Hepatocytes were obtained from the liver tissue of mandarin fish. The liver tissue was cut into small pieces and digested with trypsin (Gibco, USA). Tissue was dispersed into cells through cell strainer (Biosharp, China). Red cells were lysed with red cell lysis buffer (Biosharp, China). Cells were cultured with M199 (10% fetal bovine serum, penicillin-streptomycin solution 1‰) (Gibco, USA). The inhibitor of SET1DB chaetocin (17 (link)) (Selleck, USA) was used to treat the cells at a concentration of 3 × 10⁻⁵ mol/L for 17 h, and then the levels of H3K4me3 and pepck mRNA expression were examined with six biological replicates and three technical replicates.

You J.J., Ren P., He S., Liang X.F., Xiao Q.Q, & Zhang Y.P. (2020). Histone Methylation of H3K4 Involved in the Anorexia of Carnivorous Mandarin Fish (Siniperca chuatsi) After Feeding on a Carbohydrate-Rich Diet. Frontiers in Endocrinology, 11, 323.

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