DNA fragmentation of MDA-MB 231 breast cancer cells analysed in 1% agarose gel after 24 hours incubation with different concentration of Artonin E.

<p>Lanes 1–3 represents 3, 10 and 30 μM of Artonin E, lane 4 is untreated cancer cells, lane 5 is positive control well treated with 4μg/mL camptothecin and lane 6 is a 1kilobase DNA ladder.</p

Representative histogram analysis of the annexin V-FITC in flow cytometry assay in MDA-MD-231 cells after 24-hour and 48 hour treatment with Artonin E.

<p>Cells population in lower left quadrant are viable, in lower right quadrant are cells at early apoptosis, in upper right quadrant at late stage of apoptosis, and in upper left corner are cells at the necrotic stage.</p

Acridine orange and propidium iodide double staining of MDA-MB 231 breast cancer cells after 24 hour exposure.

<p>(A) Control, (B) 3 μM Artonin E. (C) 10 μM Artonin E, (D) 30 μM Artonin E and (E) Quantification of apoptotic morphology at 24 and 48 hours. Each result is presented as mean ± SD of three replicates. * indicates significant difference from the control of each phase (p<0.05). VC = Viable cells; BL = Cell membrane blebbing; CC = chromatin condensation; EA = Early apoptosis; LA = late apoptosis; MN = marginated nuclear chromatin; SN = secondary necrosis. Magnification X200.</p

Western blot analysis of Artonin E (3, 10, and 30 μM) treated MDA-MB231 breast cancer cells after 24 hours.

<p>(A) and the levels of protein expression quantified from the western blotting analysis of Artonin E treated MDA-MB 231 cells using Bio-rad Image Lab software (B).</p

The average half-maximal inhibitory concentrations (IC₅₀) of Artonin E, and standard agents, Tamoxifen and Paclitaxel on MDA-MB 231 and MCF-10A cell lines.

<p>The average half-maximal inhibitory concentrations (IC₅₀) of Artonin E, and standard agents, Tamoxifen and Paclitaxel on MDA-MB 231 and MCF-10A cell lines.</p

The accession number and sequence of the primers used in the gene expression studies.

<p>The accession number and sequence of the primers used in the gene expression studies.</p

MDA-MB 231 breast cancer cell cycle regulation after 12 hours treatment with (B) 3 μM, (C) 10 μM, and (D) 30 μM Artonin E.

<p>(A) is untreated control. (E) Analysis of cell population in the cycle phases. Values are mean ± standard deviation of three replicates. *Means in each cell cycle phase significantly (p<0.05) different from the control.</p

Mechanism of action of Artonin E in MDA-MB 231 breast cancer cells.

<p>Mechanism of action of Artonin E in MDA-MB 231 breast cancer cells.</p

Total ROS production by MDA-MB 231 cells after treatment with Artonin E.

<p>(A) Untreated control and (B), (C) and (D) are treatment with 3, 10 and 30 μM Artonin E, respectively. (E) is the analysis of the flow cytometric data. Values are means ± standard deviation of three replicates. *Means differ significantly (p<0.05) from that of untreated control. M1 = non-ROS while M2 indicate the percentage of cells with an increase production of ROS.</p

The percentage of MDA-MB-231 cell line viability after treatment with Artonin E.

<p>The experiment was done in triplicate at 24, 48, and 72 hours and each point are presented as mean ± SD.</p