Induction of caspases-mediated apoptosis by carnosol in the MDA-MB-231 cells.

<p>(<b>A</b>) Carnosol induced apoptosis in the MDA-MB-231 cells. Annexin V binding was carried out using Annexin V & Dead Cell kit (Millipore). Cells were treated with DMSO or various concentration of carnosol for 24 h. Detached and adherent cells were collected and stained with Annexin V and 7-AAD and then the events for early and late apoptotic cells were counted with the Muse Cell Analyzer as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109630#s2" target="_blank">Materials and Methods</a>. (<b>B</b>) Western blot analysis of caspase 3, -9, and -8 activation, PARP cleavage and Bcl1 and Bax expression in MDA-MB-231 cells were treated with increasing concentrations of carnosol (25, 50 and 100 µM) for 24 h. (<b>C</b>) Carnosol induces the depolarization of mitochondrial membrane. Mitochondrial membrane potential (MMP) was assessed with the Muse Cell Analyzer using the Muse MitoPotential kit as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109630#s2" target="_blank">Materials and Methods</a>. Data represent the mean ±SEM of at least 3 independent experiments. Student's t test was performed to determine the significance (*<i>p</i><0.05, **<i>p</i><0.01 and ***<i>p</i><0.005).