Amb4269951

Cells were plated and cultured on 24-well plates. Amb4269951 and ceramide (C2 ceramide, Cayman Chemical, Ann Arbor, MI, USA) were added, and the final volume of medium in each well was 1.0 mL. C2 ceramide is a biologically active, cell permeable, and less hydrophobic analog of natural ceramides. Cell counts were measured using CellTiter-Glo^® Luminescent Cell Viability Assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions. Luminescence was measured on a FilterMax F5 Multi-Mode Microplate Reader (Molecular Devices, LLC, Sunnyvale, CA, USA).

The cell-viability assay was performed as previously described [8 (link),9 (link),10 (link),20 (link)]. MIA PaCa-2 cells were cultured in 96-well plates with 1000 cells/well. Amb4269951, Amb4269675, and ceramide (Cayman Chemical, Ann Arbor, MI, USA) were added 24 h after cell culture. The final concentration of DMSO used as solvent was set at 1%. Cell numbers were measured using a CellTiter-Glo^® Luminescent Cell Viability Assay (Promega, Madison, WI, USA). Chemiluminescence measurements were performed using a FilterMax^TM F5 Multi-Mode Microplate Reader (Molecular Devices, LLC, Sunnyvale, CA, USA).