Fig 1.
EMBS exposure results in oxidative stress.
(A) Flow cytometry of 2,7-dichlorofluorescein diacetate- and hydroethidine-stained cells to determine the influence of EMBS (0.4 μM, 24 h) exposure on hydrogen peroxide and superoxide production in MCF-7-, MDA-MB-231- and MCF-12A cells. Histograms were gated for positive- and negative staining and the percentage positive cells are included in each graph. (B) MDA-MB-231 cells were exposed to 0.4 μM EMBS for the indicated timepoints. Hydrogen peroxide (dark grey line) and superoxide (light grey line) were measured. The graph represents the average of 3 independent experiments with error bars representing s.e.m. Values represent the fold-change compared to cells at 0 h. An asterisk (*) demonstrates a statistically significant P value <0.05 when compared to vehicle-treated cells.
Fig 2.
EMBS-treated cells undergo apoptosis correlated with decreased cell viability, metabolic activity and mitochondrial membrane potential.
(A) MDA-MB-231 cells were exposed to 0.4 μM EMBS for the indicated timepoints and stained with crystal violet. The graph represents the average of three independent experiments with error bars representing s.e.m. Values represent the absorbance measurements at 570 nm. An * demonstrates a statistically significant P value <0.05 when compared to vehicle-treated cells. (B) MDA-MB-231 cells were exposed to 0.4 μM EMBS for the indicated timepoints. Subsequently, Mitotracker was quantified using flow cytometry. The graph represents three independent experiments with error bars of s.e.m. Apoptosis was measured relative to vehicle-treated cells. An * demonstrates a statistically significant P value <0.05 when compared to vehicle-treated cells. (C) Annexin V/PI staining was used to determine the number of viable (▲), necrotic (X), early- (♦) and late apoptotic cells (■) at different timepoints after EMBS exposure. The graph represents the average of three independent experiments with error bars representing s.e.m. An * demonstrates a statistically significant P value <0.05 when compared to vehicle-treated cells. (D) To measure metabolic activity cells were exposed to EMBS and simultaneously incubated with Alamar blue. At indicated timepoints Alamar Blue was measured and the percentage reduction in exposed cells was calculated relative to that in control cells. The graph represents the average of three independent experiments with error bars representing s.e.m. An * demonstrates a statistically significant P value <0.05 when compared to vehicle-treated cells.
Fig 3.
Antiproliferative and apoptotic effects exerted by EMBS are ROS-dependent.
(A) Hydrogen peroxide production of EMBS-treated cells in the presence of absence of 20 mM NAC. The graph represents the average of 3 independent experiments with error bars representing s.e.m. Values represent fold-change compared to vehicle-treated cells. A * demonstrates a statistically significant P value <0.05 when compared to EMBS-treated cells. (B) Crystal violet staining demonstrating the dose-dependent inhibition of NAC on the antiproliferative activity of 0.4 μM EMBS measured after 24 h. Cell growth is expressed as a percentage compared to cells propagated in control growth medium. The graph represents the average of 3 independent experiments with error bars representing s.e.m. An * demonstrates a statistically significant P value <0.05 when compared to EMBS-treated cells. (C) Mitochondrial membrane potential of EMBS-treated cells in the presence or absence of 20 mM NAC. Cells treated with EMBS alone (dark grey bar) or EMBS and NAC (light grey bar) were analysed using Mitotracker. The graph represents three independent experiments with error bars of s.e.m. An * demonstrates a statistically significant P value <0.05 when compared to cells exposed to 0.4 μM EMBS and 20 mM NAC. (D) MDA-MB-231 cells were either left unblocked or were blocked in G1/S by thymidine and were exposed EMBS alone or together with 20 mM NAC. The graph represents the average results of crystal violet of three independent experiments with error bars representing s.e.m. * demonstrates a statistically significant P value of <0.05 when compared to EMBS-treated cells. An ** demonstrates a statistically significant P value <0.05 when compared to cells treated with EMBS, thymidine and NAC.
Fig 4.
EMBS induces ROS/JNK-dependent apoptosis, G2/M block and endoreplication.
(A) Cell cycle progression was analysed using PI in cells treated with EMBS alone, EMBS together with NAC or EMBS together with the JNK inhibitor, SP600125. The graph represents three independent experiments showing the percentage of cells in each of five categories. Error bars represent s.e.m. An * demonstrates a statistically significant P value of <0.05 when compared to vehicle-treated cells. (B) Cell cycle progression analysis of analysis of EMBS-treated MDA-MB-231 cells in the presence or absence of NAC after 4 h, 24h and 48 h. The graph represents three independent experiments showing percentage of cells in each of four categories. (C) Cell cycle progression analysis of analysis of EMBS-treated MDA-MB-231 cells in the presence or absence of Jnk inhibitor (SP600125) after 4 h, 24 h and 48 h. The graph represents three independent experiments showing percentage of cells in each of four categories. (D) Flow cytometry was conducted to investigate if JNK inhibition influences EMBS-induced ROS production. Cells were either treated with 0.4 μM EMBS for 24 h in the presence or absence of the JNK inhibitor (SP600125) after which DCF fluorescence was measured. The graph represents the average fold change between treated and vehicle-treated cells of 3 independent experiments with error bars representing s.e.m.
Fig 5.
EMBS induces DNA double strand breaks and endoreduplication.
(A) Confocal images of cells treated for 4 h with 0.4 μM EMBS or EMBS and 20 mM NAC were quantified by determining pixel values for H2A staining divided by the number of cells per image. The average of 100 cells per treatment was used and the average of three independent experiments are represented with s.e.m. represented by the error bars. A significant difference between the DMSO treated and EMBS treated cells was observed at a P-value of 0.25. (B) Cells incubated with EMBS or EMBS and NAC for 24 h were stained for phosphorylated H2A (red) and DAPI (blue). Representative images show an increase in the number of deformed nuclei in EMBS treated cells, while these were mostly absent in control or NAC-treated cells.
Fig 6.
EMBS does not induce autophagy.
(A) Autophagy was measured by determining the levels of LC3B II by western blot. Cells were treated with DMSO as control (lane 1), bafilomycin A1 (lane 2) as positive control or EMBS for 4 (lane 3), 10 (lane 4), 14 (lane 5) or 24 (lane 6) h. Total protein loading was measured by western blot against actin. The graph represents expression as a percentage of the positive control that represents cells exposed to bafilomycin A1. Both blot and graph are representative of three independent experiments.