Cell viability assay For each experiment, cells were seeded in duplicate 96 well white walled plates at 4,000 cells per well. After over night incubation, cells were treated with combinations of DMSO as vehicle control, MK 1775, and MK 8776 for 72 hours. Cell viability was determined by measuring ATP with Vialight Plus according to manufacturers instructions. Drug meantime potency was calculated as the ratio of relative light units in compound treated wells over DMSO treated control wells and expressed as % DMSO control. Compound EC50s were calculated in GraphPad Prism using a 4 parameter variable slope sigmoidal dose response curve fit. Flow cytometry Cells were treated with indicated concentrations of MK 1775, MK 8776, both, or an equivalent volume of vehicle for a fixed time period.
At time of harvest, cells were counted and then fixed in ice cold 70% ethanol overnight before staining with anti phospho histone H2AX antibody conjugated to FITC, anti phospho histone H3 antibody conjugated to Alexa FluorW 647, and propidium iodide. Samples were read on the LSR II flow cytometer, and data were analyzed using FlowJo software version 7. 5. 5. Animals and xenograft studies CD 1 Nu Nu female mice aged 5 6 weeks were obtained from Charles River Laboratories and housed in our animal care facility at standard laboratory conditions and fed 2018S autoclaveable diet and water ad libitum. The proto col was approved by Mercks Institutional Animal Care and Use Committee. Mice were inoculated with 5 x 106 LoVo cells in 100 uL subcutaneously into the right flank.
When tumor volume reached 200 mm3 mice were pair matched so each group had a similar mean and standard deviation. Tumor vol ume and body weights were recorded bi weekly. Mice received 4 treatment cycles of twice daily dosing for 2 days receiving either vehicle, MK 1775, and or MK 8776 For pharmacodynamic assays, mice were dosed with 60 mpk of each compound. In vivo pharmacodynamic assays Xenograft tumors were fixed in 10% formalin, paraffin embedded and sectioned at 5 um. Tumor sections were immunostained with rabbit monoclonal anti phospho CHK1 antibody, rabbit polyclonal anti gamma histone H2AX antibody, rabbit polyclonal anti phospho CDC2 Y15 antibody and rabbit monoclonal anti Ki67 antibody. Labeled antigens were visualized using Omni Map anti rabbit HRP and peroxidase substrate.
Slides were digitized using an Aperio ScanScope XT Image Sys tem and immunostained cells were quantified using Aperio Imagescope software. The percentage of cells showing immunostaining in Entinostat each tumor was calculated relative to the number of total cells with necrotic regions excluded. Bliss synergy calculations The Bliss independence model is used to define the ef fect of two drugs assumed to act through independent mechanisms.