Proteome profiler apoptosis array

Cell lysates were obtained from A375 cells, after 72hrs post-treatment. The expression of 35 apoptosis-related proteins was analysed using the antibody-based Proteome Profiler™ Apoptosis Array from R&D Systems (Cat No. ARY009) as per manufacturer's instructions.

Approximately 5 X 10⁵ cells were seeded in 6-well plates for 24 h. Then, cells were treated with an IC₅₀ concentration of D14 and C22 compounds and vehicle for 24 h. Cells were harvested with 0.25% trypsin, washed with phosphate buffered saline (PBS), and collected together by centrifugation. Apoptosis was determined using the Apoptosis/Necrosis Detection kit (Abcam, catalog No. ab176749, Cambridge, England) according to the manufacturer’s instructions and analyzed by flow cytometry using a FACSCalibur instrument (BD Biosciences), followed by data analysis using FlowJo software (Tree Star Inc). All experiments were performed in triplicate. Proteome Profiler Apoptosis Array (R&D Systems: ARY009) was used to evaluate the activity of D14 and C22 compounds on MIA-PaCa-2 cancer cells to determine the signaling pathways associated with cell death via Kras4B inhibition, which were done following the manufacturer’s instructions.