FLLL32 induced caspase-dependent apoptosis and loss of mitochondrial membrane potential. (A) Processing of caspase proteins was measured by immunoblot following a 24 hour treatment of A375 cells with FLLL32. The molecular weight of each pro-caspase and their active forms are listed on each blot in kilodaltons (kDa). Data shown are representative of at least two independent experiments. (B) A375 cells were treated for 24 hours with FLLL32 and stained with 150 nM TMRE to assay loss of mitochondrial membrane potential (ΔΨm) by flow cytometry. Voltage was set using unstained cells (M1). The percentage of cells with reduced ΔΨm is denoted above each histogram. Data are representative of three or more independent experiments. Flow cytometric analysis of annexin V/PI staining following a 48 hour treatment of (C) A375 cells with FLLL32 in the presence of the Z VAD-FMK pan-caspase inhibitor or the Z-FA-FMK control compound. Inhibitors were used at 50μM and the percentage of cells in each quadrant are shown. (D) Immunoblot analysis of A375 cells following a 48 hour treatment with FLLL32 cultured in the presence of the Z-VAD-FMK pan caspase inhibitor (+) or the Z FA FMK control compound (-). Data shown are representative of two separate experiments with the A375 cell line and were also reproducible in the Hs294T cell line (Additional File 1: Figure S2).