Inhibition of [³H-methyl]thymidine incorporation by HMI-1a3 and PMA.

<p>HeLa cells were treated for 6 or 24 h with increasing concentrations of HMI-1a3 (A) or PMA (B), and the incorporation of thymidine was determined. Error bars indicate SEM from 3 independent experiments.</p

Effects of C1 domain ligands on HeLa cell viability.

<p>Cells were exposed to isophthalate derivatives (A) and PMA or bryostatin 1 (B) for 24 h, and cell viability was determined by MTT assay. Results are expressed as mean + SEM (n = 3–6; n = 2 for the HMI-24a group).</p

Effects of isophthalate derivatives and PMA on HeLa cell proliferation.

<p>HeLa cells were treated with test compounds and photographed automatically for 72 h with Cell-IQ®. Cell numbers were quantified from each image using Cell-IQ Analyzer® software. A, normalized total cell number at 72 h is expressed as a percentage of control. Results represent mean + SEM from 3–5 independent experiments except for NI-15e where n = 2. B and C, proliferation of HeLa cells during the 72-h experiment is shown as normalized total cell number in untreated wells and after treatment with different concentrations of HMI-1a3 and HMP-27 (B) and PMA (C). Cell numbers shown were determined from images taken with 3-h intervals from a single, representative experiment. Error bars correspond to SEM of 4 images taken from different positions within the same well. Experiments were repeated at least 3 times with similar results.</p

Effect of isophthalate derivatives on HeLa cell morphology.

<p>Untreated cells (A, D, G) and cells treated with 20 µM of HMP-27 (B, E, H) or 20 µM of HMI-1a3 (C, F, I) were imaged automatically for 72 h with Cell-IQ® and analyzed using Cell-IQ Analyzer® software. Representative photomicrographs taken at time points 0 h (A–C), 24 h (D–F) and 72 h (G–I) are presented. J–M, quantification results from a single, representative experiment (mean from 4 images in different positions within the same well) of untreated cells (J) and cells treated with 20 µM of HMP-27 (K), 20 µM of HMI-1a3 (L) or 20 µM of HMI-1a3 and 40 µM of BAF. Experiments were repeated at least 3 times with similar results.</p

Effects of C1 domain ligands on the expression of cell cycle markers.

<p>HeLa cells were exposed to compounds for 24 h and proteins were detected with Western blotting. A, representative Western blots showing specific immunoreactive bands after various treatments (lanes 1–6) as follows: lane 1, untreated cells; lane 2, 0.2% DMSO; lane 3, PMA at 100 nM; lane 4, bryostatin 1 at 100 nM; lane 5, HMI-1a3 at 10 µM; lane 6, HMI-1a3 at 20 µM. B, quantification results of Western blots expressed as mean + SEM (n = 3-4). OD = optical density.</p

Analytical conditions in the separation and quantification of glucuronides.

1<p>LOD, limit of detection; LOQ, limit of quantification; values are calculated assuming maximal injection volume;</p>2<p>The UV signal for was correlated with fluoresence for enhanced sensitivity.</p

Enzyme kinetics of UGT2B15-catalyzed glucuronidation of 17α-estradiol (A) and 4-MU (B), in the absence and presence of BSA.

<p>The reaction with 17α-estradiol was analyzed for the formation of 17α-estradiol-3-β-D-glucuronide. The glucuronidation rates are presented as the average value ± S.E. These values are not expression normalized because we used commercial UGT2B15 without appropriate His-tag. The concentrations of substrates were corrected for binding to 0.1% BSA. The determined enzyme kinetic parameters are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054767#pone-0054767-t003" target="_blank">Table 3</a>. See <i>Materials and Methods</i> for all further details.</p

Enzyme kinetics of UGT2B7-catalyzed glucuronidation of 17α-estradiol (A), 17β-estradiol (B), and 4-MU (C), in the absence and presence of BSA.

<p>The reactions with 17α-estradiol and 17β-estradiol were analyzed for the formation of 17α-estradiol-17-β-D-glucuronide and 17β-estradiol-17-β-D-glucuronide, respectively. The glucuronidation rates are presented as the average value ± S.E., and are expression level-normalized values. The concentrations of substrates were corrected for binding to 0.1% BSA. The determined enzyme kinetic parameters are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054767#pone-0054767-t003" target="_blank">Table 3</a>. See <i>Materials and Methods</i> for all further details.</p

The chemical structures of the substrates that were used in this study.

<p>In 17α- and 17β-estradiol both the 3-OH and the 17-OH can be conjugated, mostly by different UGTs (see Figs. 2 and 3). In the case of entacapone, the glucuronidation occurs on hydroxy group in position 3.</p

Structures and binding affinities of isophthalate derivatives.

<p>Binding affinity is expressed as mean ± SEM (n = 3–8) of the inhibition percentage of [³H]phorbol-12,13-dibutyrate ([³H]PDBu) binding at compound concentrations of 20 µM. Binding data are from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0020053#pone.0020053-BoijeafGenns2" target="_blank">[14]</a> and are reprinted with permission from the American Chemical Society.</p