LY3009120 in escalating dose cohorts given orally BID (twice daily) every 12 hours for a 28-day cycle

A375 cells (catalog no. CRL-1619) were obtained from the American Type Culture Collection (ATCC, Manassas, VA). Cells were grown in DMEM high glucose supplemented with 10% characterized fetal bovine serum (Invitrogen, Carlsbad, CA) and 1% penicillin/streptomycin/L-glutamine at 37 °C, 5% CO2, and 95% humidity. Cells were allowed to expand until 70−95% confluency at which point they were subcultured or harvested for assay use. A serial dilution of test compound (LY-3009120) was dispensed into a 384-well black clear bottom plate in triplicate. Six-hundred-twenty-five cells were added per well in 50 μL of complete growth medium in the 384-well plate. Plates were incubated for 67 h at 37 °C, 5% CO2, and 95% humidity. At the end of the incubation period, 10 μL of a 440 μM solution of resazurin (Sigma, St. Louis, MO) in PBS was added to each well of the plate and plates were incubated for an additional 5 h at 37 °C, 5% CO2, and 95% humidity. Plates were read on a Synergy2 reader (Biotek, Winooski, VT) using an excitation of 540 nm and an emission of 600 nm.