Cell culture products

cDNA containing the complete coding region of the hMT₁ (human Mel_1a cloned into pcDNAI) and hMT₂ melatonin receptor (human Mel_1b cloned into pcDNA-3) were provided by Drs. S. M. Reppert and D. Weaver (Department of Neurobiology, University of Massachusetts Medical School, Worcester, MA, USA).^{10 (link),38 (link)} Effectene transfection kit was obtained from Qiagen (Valencia, CA, USA). Cell culture products were obtained from Life Technologies (Carlsbad, CA, USA) and Corning (Manassas, VA, USA). 2-[¹²⁵I]-Iodomelatonin (SA: 2200 Ci (81.4TBq)/mmol) was purchased from PerkinElmer (Waltham, MA, USA). Carbaryl and carbofuran were obtained from Sigma-Aldrich (St. Louis, MO, USA).
Carbaryl (13 mM stock solution) was dissolved in ethanol, and carbofuran (13 mM stock solution) was dissolved in dimethyl sulfoxide (DMSO). Carbaryl and carbofuran were next diluted to 1.3 mM in 50% ethanol/50% Tris-HCl buffer (50 mM and 10 mM MgCl₂ at pH 7.4 and 25 °C). Further dilutions were performed in Tris-HCl buffer. The ethanol or DMSO concentration in assays with 1 mM, 0.1 mM, or 0.01 mM carbamate compounds was 8%, 4%, or 0.4% respectively, producing minimal inhibition of 2-[¹²⁵I]-iodomelatonin binding when tested alone.

293T cells were cultured in DMEM (high glucose) medium containing 10% FBS. K562 cells were cultured in IMDM medium plus 10% FBS. Enriched fresh hematopoietic stem and progenitor cells (HSPCs) were cultured in vitro in Stemspan (STEMCELL Technologies) supplemented with 10 μg/ml heparin (Sigma), 10 ng/ml mouse SCF, 20 ng/ml mouse TPO, 20 ng/ml human IGF-II, 10 ng/ml mouse FGF-1, and 100 ng/ml human Angptl3 ²⁵ . All recombinant proteins were purchased from PEPROTECH or PROSPEC companies. All cell culture products were purchased from Life Technologies unless otherwise mentioned. All small molecule inhibitors were purchased from Cayman Chemicals.