Tritonx

PDX cells (3 × 10⁶) were plated in a 6-well plate and treated with 10 or 25 μM of 6-Me for 72 hours. Cells were separated into a single cell suspension using Accutase® (Corning), washed with phosphate-buffered saline (PBS), and fixed with 100% ethanol at 4 °C for at least 30 minutes. Following a second wash with PBS, cells were stained with a propidium iodide (Invitrogen, Carlsbad, CA) solution containing 0.1% TritonX (Active Motif, Carlsbad, CA), and 0.1 mg/mL RNAse A (Qiagen, Germantown, MD). Cell-cycle data were obtained using the Attune™ NxT Flow Cytometer (Invitrogen™, Thermo Fisher, Eugene, OR) and analyzed using the FlowJo software (FlowJo, LLC, Ashland, OR).

To evaluate cell cycle, HuH6 cells (5 × 10⁵) were synchronized overnight in media with 4% FBS. HuH6 cells were treated with 3364 (6 μM) or 8385 (8 μM) for 24 h in standard media with 10% FBS. COA67 cells (3 × 10⁶) were plated and treated with 3364 or 8385 (4 μM) and treated for 24 h. Cells were washed with phosphate-buffered saline (PBS) and fixed on ice for 30 min with 100% ethanol. Following a second PBS wash, cells were stained with 200 μL of propidium iodide (PI, Invitrogen, Waltham, MA), RNAse A (0.1 mg/mL, Qiagen, Germantown, MD), and 0.1% TritonX (Active Motif, Carlsbad, CA). FACSCalibur^™ Flow Cytometer (BD Biosciences, Franklin Lakes, NJ) was used for evaluation and data analysis completed with FlowJo software (FlowJo, LLC, Ashland, OR). Experiments were repeated with three biologic replicates and data reported as mean percentage cells in phase ± SEM.