Hotspots of aberrant enhancer activity in fibrolamellar carcinoma reveal molecular mechanisms of oncogenesis and intrinsic drug resistance

Timothy A. Dinh; Ramja Sritharan; F. Donelson Smith; Adam B. Francisco; Rosanna K. Ma; Rodica P. Bunaciu; Matt Kanke; Charles G. Danko; Andrew P. Massa; John D. Scott; Praveen Sethupathy

For AML12 cells seeded in 96-well plates, a series of ½-log unit dilutions of cobimetinib (10 mM stock in DMSO) were made in DMSO at 1000X final desired concentrations. From these stocks, 1:200 dilutions were made in fresh AML12 media. 30 µl of these starting dilutions was added to appropriate wells using a multi-channel pipettor. This results in a further 1:5 dilution and a final 1:1000 dilution with a final volume of 150 µl per well. Outer plate wells were filled with media and a no-cells/no-treatment set of wells was included for background. Cell plates were grown for a further 4-5 days. FLC cells were seeded in 96-well plates at a density of 3200 cells/well in a volume of 100 µl per well for CellTiter-Glo (Promega, Madison, WI) assays. The following day, 2X concentrations of drug or DMSO were prepared. 50 µl of media was removed from each well and 50 µl of 2X drug or vehicle (final concentration 1X) was added to each well. Cells were incubated for 48 hours before assessing cell viability.