Gnf 2

Roswell Park Memorial Institute (RPMI) 1640 medium, Dulbecco's modified Eagle's medium (DMEM), Dulbecco's phosphate-buffered saline (DPBS), trypsin, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), non-essential amino acids (NEAA), sodium pyruvate, blasticidin, penicillin/streptomycin, and CCF₄ reagents were purchased from Life Technologies. Defined and dialyzed fetal bovine serum (FBS) was acquired from Thermo Fisher Scientific. ME-180 cell line, McCoy's 5A medium, and FBS were purchased from American Type Culture Collection (ATCC). Nano-Glo, CellTiter Blue, CellTiter Glo reagents were acquired from Promega. The NCATS Pharmaceutical Collection (NPC) library [8 ] was prepared as stock solutions in DMSO in 1536-well plates, where each plate corresponds to an indicated concentration of test compounds. G418 was purchased from Sigma-Aldrich. Azacitidine, AZD5363, bortezomib, camptothecin, enzastaurin, GDC-0994, GNF- 2, mitoxantrone, mycophenolate mofetil, niclosamide, OSI-906, PHT-427, PI-103, PP-242, selumetinib, TAK-632, trametinib, topotecan, volasertib, and YC-1 were purchased from SelleckChem. Cylcoheximide was purchased from Tocris Bioscience. The compound purity and identity were confirmed by quality control analysis at NCATS.

The pGEX‐4T‐1‐rRANKL vector was a generous gift from Dr. Steven Teitelbaum that encodes the GST fusion construct containing the biologically active extracellular domain of RANKL (GST‐RANKL).30, 31 The plasmid was transformed into and expressed in Origami B(DE3) cells (EMD Millipore, Billerica, MA, USA) that co‐expresses chaperone proteins and was generously provided by Dr. Ding Xu. The GST‐RANKL fusion protein was purified using a BioScale Mini Profinity GST cartridge (Bio‐Rad, Hercules, CA, USA) and a subsequent size‐exclusion chromatography step. Endotoxins were removed using the Pierce High Capacity Endotoxin Removal Resin (Thermo Fisher Scientific, Waltham, MA, USA). GNF‐2 was obtained from Selleck Chemicals (Houston, TX, USA).