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

Cell viability was assessed by CellTiter-Glo or crystal violet staining. For CellTiter-Glo, 96-well plates were removed from incubator and placed at room temperature for 30 minutes to equilibrate. Room temperature CellTiter-Glo reagent was added and cells were shaken for 2 minutes. Plates were then incubated for 10 min at room temperature. Luminescence was measured using a POLARStar Omega plate reader (BMG LabTech, Ortenberg, Germany; Em Filter -empty; Gain = 3600, orbital averaging ON, diameter = 5, cycles = 6) or a Synergy 2 Microplate Reader (Biotek, Winooski, VT; area scan; integration time = 0.50 seconds). For crystal violet staining, AML12 cells were rinsed in PBS and fixed in 4% paraformaldehyde in PBS for 20 minutes. Two water washes were performed and cells were stained with 0.25% crystal violet in 10% methanol for 20 minutes. Finally, three water washes were performed and plates were allowed to dry at room temperature for at least 24 hours. Images were captured with a custom digital photography set-up on a Canon 5D, MkI with a Sigma 150-600 mm lens. To quantify crystal violet staining, dye was dissolved in 10% acetic acid (300 µl per well). An aliquot was removed to a clear 96-well plate and A590 absorbance was measured using a POLARStar Omega plate reader (BMG LabTech). Signal was kept in the linear range by 1:2 -1:4 dilution with 10% acetic acid where necessary.