Figure 4

The effects of TXS inhibition on tumour cell apoptosis. A) Apoptosis was induced in a dose-dependant manner following 24 h treatment with ozagrel, relative to untreated control cells. Cell health following ozagrel treatment was assessed using 3 spectrally distinct flourophores to examine nuclei, f-actin (marker of cytoskeletal integrity), and mitochondrial mass/potential. Reduced f-actin levels demonstrate a loss in cellular integrity during apoptosis. Membrane blebbing also occurs and mitochondrial activity occurs, coupled with a loss of potential across the mitochondrial membrane. These markers were quantified by the Kinetic Scan HCS reader and are represented (B). Similar observations were made in SKMES-1 cells (data not shown). Apoptosis was confirmed following selective TXS inhibition by Cell Death Detection ELISA and DNA laddering in both cell lines (A-549 shown as representative). Cell Death ELISA demonstrated increased apoptosis in a concentration dependant manner, with fold induction expressed as a ratio of control cells (C). DNA laddering was also observed following ozagrel treatment at both 500 nM and 5 μM concentrations (D).