Inhibition of colony growth by <i>O. majorana</i> extract.

<p>Inhibition of colony growth was assessed by measuring the size of the colonies obtained in vehicle (ethanol)- and OME-treated plates. Data were compared with those obtained for the 2 weeks colonies. Two types of colonies were counted and depending on their diameter were categorized as large (≥200 µm) and small (<200−≥50 µm).</p

Differential regulation of survivin expression by <i>OME</i> in MDA-MB-231 cells.

<p>Western blot analysis showing a differential effect on survivin expression by different concentrations of OME in MDA-MB-231 cells. Whole cell protein were extracted from OME or vehicle (ethanol)-treated cells and subjected to Western blot analysis, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056649#s2" target="_blank">Materials and Methods</a>, for survivin and β-actin (loading control) proteins.</p

Induction of G2/M cell cycle arrest and apoptosis by <i>O. majorana</i> extract in MDA-MB-231 cells.

<p>(A) MDA-MB-231 cells (1.8×10⁶) seeded on 100 mm culture dish were exposed various concentrations of <i>O. majorana</i> extract or equal volume of vehicle (ethanol) as control for 24 h. Following treatment, cells were harvested, fixed, stained with propidium iodide, and analyzed for cell cycle distribution by flow cytometry. Data represent the mean of three independent experiments. The percentage of cells in sub-G1 (apoptosis), G1, S and G2/M appears at the upper right of each graph. (B) Expression of cell cycle regulator in OME-treated MDA-MB-231. Western blot analysis of phospho(ser10)-H3, and cyclin B1 in MDA-MB231 cells exposed for 24 h to ethanol or indicated concentrations of OME. (C) Stimulation of caspase 3/7 activity in MDA-MB-231 cells after exposure to OME (0–600 µg/mL) for 24 h and 48 h, relative to a similar amount of viable ethanol-treated cells. The relative caspase 3/7 activity was normalized to the number of viable cells per well and is expressed as fold of induction compared to the control. (D) Concentration-dependent induction of PARP cleavage in OME-treated MDA-MB231 cells. Cells were treated with or without increasing concentrations of the extract and proteins were extracted as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056649#s2" target="_blank">Materials and Methods</a>. Western blot analysis was carried out using anti-PARP antibodies. (*<i>p</i><0.05, **<i>p</i><0.005 and ***<i>p</i><0.0005).</p

The proposed signal pathways on <i>O. majorana-</i>induced cell cycle arrest, at low concentrations, and apoptosis, at high concentrations, in the triple negative mutant p53 human breast cancer MDA-MB-231 cells.

<p>The proposed signal pathways on <i>O. majorana-</i>induced cell cycle arrest, at low concentrations, and apoptosis, at high concentrations, in the triple negative mutant p53 human breast cancer MDA-MB-231 cells.</p

<i>O. majorana</i> extract induces a dose-dependent activation of γH2AX, a marker of DNA double-strands breaks, in MDA-MB 231 cells.

<p>(A) Western blot analysis of phosphor-H2AX (ser 139) in MDA-MB231 cells exposed for 6 and 24h with the indicated concentrations of OME or equal volume of vehicle (ethanol) as control. (B) Immunofluorescence staining for γH2AX in OME-treated MDA-MB 231cells. Cells were treated with vehicle or 150, 300 and 450 µg/mL extract for 24 h, fixed, permeabilized, and then processed for immunofluorescence using antibodies against p-H2AX (ser 139) protein. DAPI was used as a nuclear stain.</p

<i>O. majorana</i> induces apoptosis by activation of caspase 8 and upregulation of TNF-α.

<p>(A) <i>O. majorana</i> extract induces an activation of caspase 8 but not caspase 9 in MDA-MB-231 cells. MDA-MB-231 cells were incubated with various concentrations of the extract for 24 h. The caspase activation induced by the OME was assayed as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056649#s2" target="_blank">Materials and methods</a>. The relative caspase 8 and 9 activity was normalized to the number of viable cells per well and is expressed as fold of induction compared to the control. (B) Western blot analysis showing an increase in cellular TNF-α protein in the MDA-MB-231 cells treated with OME. Whole cell protein were extracted from OME-treated cells or vehicle (ethanol)-treated cells and subjected to Western blot analysis, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056649#s2" target="_blank">Materials and Methods</a>, for TNF-α and β-actin (loading control) proteins. (C) Immunofluorescence staining for TNF-α in OME-treated MDA-MB- 231cells. Cells were treated with 150 and 300 µg/mL of the extract for 24 h, fixed, permeabilized, and then processed for immunofluorescence using antibodies against TNF-α protein. DAPI was used as a nuclear stain. (*<i>p</i><0.05 and **<i>p</i><0.005).</p

<i>O. majorana</i> induces hyperacetylation of histone H3 (AcH3) and H4 (AcH4).

<p>(A) Protein levels of Ac-H3 and Ac-H4, extracted from OME-treated cells, were detected by western blot using antibodies specific for the modified histone (Ac-H3) and Ac-H4. β-actin was used as loading control. (B) Immunofluorescence staining of Ac-H3 and Ac-H4 in MDA-MB231 cells treated with 150, 300 and 450 µg/mL of OME or equal volume of vehicle (ethanol) as control for 24 h. Cells were fixed, permeabilized, and then processed for immunofluorescence using antibodies against the indicated modified histones. DAPI was used as a nuclear stain.</p

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).</p

ROS generation and DNA damage accumulation in carnosol-treated MDA-MB-231 cells.

<p>(<b>A</b>) Carnosol induces ROS generation in MDA-MB-231 cells. Cells were treated with indicated concentrations of carnosol for 24 h and ROS production was analyzed, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0109630#s2" target="_blank">Material and Methods</a>, using DCFDA fluorescence stain. (<b>B</b>) time-course measurement of ROS generation in carnosol-treated MDA-MB-231 cells. Cells were treated with 100 µM carnosol and ROS generation was examined at different time-point (1, 3 and 6 h). (<b>C–D</b>) Concentration and time-dependent accumulation of γH2AX, marker of DNA damage, in carnosol-treated cells. DNA damage was evaluated by measuring, by Western blotting, the level γH2AX accumulation using anti-phospho-H2AX (ser 139) antibody.</p

Carnosol induces G2 arrest in MDA-MB-231 cells.

<p>(<b>A–B</b>) Cell cycle distribution analysis of carnosol-induced G2 cell-cycle arrest. MDA-MB 231 cells were treated with carnosol at the indicated concentrations for 24 h, then analyzed with Muse Cell Analyzer using the Muse cell cycle 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>. (<b>C–D</b>) Effect of Carnosol on cell cycle markers. Cells were treated with DMSO or indicated concentrations of carnosol and the expression level of histone H3-p(ser10), Cyclin B1, p21 and p27 was examined by western blotting after 24 post-treatment.</p