Cell cycle analysis examined using flow cytometry on HT-29 cells after 72 h treatment.

<p>(A) Cells treated with (a) DMSO at the final concentration of 0.1%. (b) FKB at a concentration of 12.5 (3.55 μg/mL), (c) 25 (7.1 μg/mL), (d) 50 μM (14.2 μg/mL), and (e) Percentage of cell cycle distribution in different phases. (B) Cells treated with (a) DMSO at the final concentration of 0.1%. (b) APN at 12.5 (3.37 μg/mL), (c) 25 (6.75 μg/mL), (d) 50 μM (13.5 μg/mL) concentrations, and (e) Percentage of cell cycle distribution in different phases. G0/G1, G2+M, and S are cell phases, respectively; subG0/G1 refers to cell death due to DNA fragmentation. Data are expressed as Mean±SD of three independent experiments, *p<0.001, ns: non-significant compared to the normal control.</p

Levels of MDM2 and p53 proteins expressed in HT-29 cells.

<p>(A) Level of proteins in cells treated with (a) crude hexane (IC₂₅: 10.52, IC₅₀: 21.05, and IC₇₅:42.1 μg/mL) and chloroform (IC₂₅: 9.5, IC₅₀: 19.09, and IC₇₅:38.18 μg/mL) extracts. (b) 25 μM (7.1 μg/ mL) of FKB at different time interval. (c) 25 μM (6.75 μg/mL) of APN at different time interval. (B) Level of MDM2 and p53 protein expression quantified from western blotting analysis using Bio-rad Image Lab software in HT 29 cells treated with (a) Hexane and chloroform extracts (b) FKB, and (c) APN. Data are expressed as Mean±SD; ns: non-significant; *p<0.05; **p<0.01; ***p<0.01; ns: non-significant compared to the DMSO control. DC: DMSO used as negative control at a final concentration of 0.1%.</p

DNA fragmentation analysed in 1% agarose gel after 72 h incubation with different concentration of either FKB or APN.

<p>MW: DNA marker; DC: DMSO treated control; all concentrations are in μM.</p

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

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

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

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

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

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

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