Caspase 3/7 assay

A Caspase 3/7 assay was used to further evaluate the apoptotic effects of guadecitabine. Miapaca-2 and Panc1 were treated with 0.14x10^-3 μM of guadecitabine for 3 days. Cells had either no rest, 5 days’ rest, or 10 days’ rest. Cells were then plated into 96 well plates at 2000 cells/well. Caspase activity was read as per the manufacturer’s instructions (Promega) on a luminometer. After measuring the luminescence signal, data for each concentration was divided by the respective control before plotting percent caspase activity, and the data was plotted using Graph Pad Prism.

Chemopriming activity of guadecitabine: the Caspase 3/7 assay was also used to measure apoptosis due to chemosensitization. Cells were again treated with guadecitabine at a concentration of 0.14x10^-3 μM for 3 days. Cells were then plated into 96 well plates (representing no rest) at 2000 cell/ well, or allowed to rest for 5 days before reseeding into a 96 well plate. Cells were allowed to adhere for 24 hours before being treated with Irinotecan (0–2 μM) for five days. Caspase activity was again measured using a Promega kit, and data analysis was preformed using methods described above.