fixation in ice-cold methanol at 220uC and were subsequently incubated with the primary antibody cocktail, 2methoxyestradiol-bis-sulphamate-treated cells, ESE-16-treated cells, ESE-15-one-treated cells and EMBS-treated cells after 24 h exposure. An increase in the number of cells presenting with compromised mitochondrial potential was demonstrated in all three compound-treated cells when compared to the vehicle-treated control cells. doi:10.1371/journal.pone.0071935.g005 detyrosinated tubulin, anti-detyrosinated antibody YL1/2, BSA 0.3%, tween 0.1%). After incubation, cells were incubated with the secondary antibodies, namely Alexa 488 and Cy3. Samples were visualized using a Zeiss AxioImager Z1 microscope controlled by Axiovision softwared . Images were captured using an Ocra R2 N/B camera and assessed for microtubule integrity and ratio of tyrosinated to detyrosinated tubulin. Statistical analysis of data. The ANOVA students’t-test was used to determine the analytical variation in experimental procedures and biological variations within each experiment. Cell growth studies were repeated three times with a sample size of 6 in each experiment. 
Means are presented in bar charts, with T-bars referring to standard deviation. A P-values of,0.05 was regarded as statistically significant. Flow cytometry results were analyzed from 10 000 events. ESE-16 statistically significantly reduced cell growth to 45.59%. Growth of EMBS-treated cells was not statistically significantly inhibited. A statistically significant decrease in cell growth was observed at a concentration of 0.5 mM with ESE16, ESE-15-one and EMBS when compared to vehicle-treated cells. Cells exposed to 5 mM and 10 mM of ESE-15-one and EMBS revealed a slight decrease in cell growth. This observed DMXB-A biological activity biphasic effect is also a characteristic of 2ME2. Cells treated with 25 mM of all compounds respectively displayed a pronounced reduction in cell growth. Cells exposed to ESE-16 demonstrated the most prominent decrease in cell growth when compared to cells exposed to ESE-15-one and EMBS. The concentration of ESE-15-one, EMBS and ESE-16 that significantly inhibits HeLa proliferation after 24 h of 15325591 exposure was found to be 0.5 mM. This concentration was thus chosen as the dose of exposure for subsequent experiments. Results Cell number determination Dose-dependent studies were conducted with the purpose of evaluating the antiproliferative effects of ESE-15-one, EMBS and ESE-16 in HeLa cells after 24 h of exposure. As previously mentioned, a dosage range of 0.125 mM was chosen since it was previously found that the compounds caused a decrease in cell growth in this concentration range. ESE-15-one statistically significantly reduced cell growth to 80.31% and 0.1 mM Cytotoxicity: Lactate dehydrogenase assay The LDH assay was conducted to measure cytotoxicity of ESE15-one, EMBS and ESE-16. A statistically insignificant increase in LDH levels was observed in the compounds-treated cells when compared to the vehicle-treated cells. ESE-16-treated cells revealed the largest increase in LDH release when compared to vehicle-treated cells and cells treated with ESE-15-one and EMBS. Sulphamoylated Analogues Induce Apoptosis Analysis of cell morphology using transmission electron microscopy Transmission electron microscopy revealed vacuoles in cells 20573509 treated with ESE-15-one, EMBS, ESE-16 and 2-methoxyestradiol-bis-sulphamate. The latter was used as a positive control for apoptosis and autophagy. Increased o
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