Perhexiline maleate

Cells were plated at a density of 2000–4000 cells/well on laminin-coated 96-well plates in a minimum of triplicates. After three days of incubation, compound was added to each well in concentrations from 0.07–20 µM and incubated at 37 °C, in 5% CO₂, 95% humidity for eight days (192 h). For Activin A / BMP4 studies, cells were supplemented with media containing BMP4 (10 ng/ml, Peprotech, London UK) or Activin A (10 ng/ml, Thermo Fisher) 24 h prior to adding the drug. Drug was added in media supplemented with Activin A and BMP4 in concentrations from 0.07 to 20 µM and incubated at 37 °C, 5% CO₂, 95% humidity for eight days (192 h). Dorsomorphin, DMH1, perhexiline maleate, LDN-193189, LDN-212854 and LDN-214117 were purchased from Sigma-Aldrich; saracatinib and momelotinib were purchased from Selleckchem (Houston, TX, USA); K02288 was purchased from BioFocus (Saffron Walden, UK); LDN-213844 (K03841b), LDN-212838 (K03449c) and K05907 were synthesised at the Structural Genomics Consortium, Oxford (see Supplementary Methods for chemical structures and synthesis). LDN-213819 was a kind gift from Paul Yu and Greg Cuny (Harvard University). Cell viability was assessed by the CellTiter-Glo luminescent cell viability assay (Promega, Madison, WI, USA) and GI₅₀ values were calculated using GraphPad Prism version 6 as the concentration of compound required to reduce cell viability by 50%.

Cell lines (LNCaP and 22Rv1) were obtained from the University of Colorado Cancer Center (UCCC) Tissue Culture Shared Resource (2014) and were authenticated by Single Tandem Repeat analysis. LNCaP MDV-resistant cells were made by growing them in increasing concentrations of MDV ranging from 1.25 to 2.5 uM for 6 months. Cells were passaged in RPMI media containing 5% FBS supplemented with amino acids and Insulin (Hyclone). TRAMPC1 cells (Gift from Dr. Agarwal, 2015, originally from ATCC) were grown in DMEM with 4 mmol/L L-glutamine, 5 μg/mL insulin, 10 nmol/L dihydrotestosterone (DHT), 5% FBS, and 5% Nu Serum (BD Biosciences). Etomoxir-HCl, Ranolazine·2HCL and Perhexiline maleate (all from Sigma-Aldrich, St. Louis, MO) were prepared as a 30, 40 and 15 mM stock solutions, respectively. Enzalutamide (MDV3100, Medivation, Inc), was dissolved as a 20 mM stock in DMSO.