A model for Cas II-gly-affected apoptotic pathways.

<p>Depicted are the pathways that may be affected by the action of Cas II-gly. Molecules are tag-name color-coded: red color corresponding to over-expression and green color to sub-expression, other colors describe intermediate states. We can notice that the intrinsic route to apoptosis is favored by <i>caspase-3</i> activation via <i>cyt C</i> over-expression, high ROS concentrations, as well as cell cycle arrest; whereas the extrinsic route to apoptosis is somehow diminished due to very low-expression of <i>pro-caspase-8</i> and <i>caspase-8</i>. Anti-apoptotic processes are switched-down due to low levels of <i>Bcl-2</i>.</p

TNF signaling is affected in HeLa cells treated with Cas II-gly favoring apoptosis rather than differentiation and proliferation.

<p>Image based in an analysis of the gene expression matrix in the Ingenuity® database for Systems Pathways Analysis (IPA) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054664#pone.0054664-Ingenuity1" target="_blank">[28]</a>. Molecules in green are transcriptionally down-regulated whereas molecules in red are transcriptionally over-expressed. Notice the abundance of molecules -such as HMOX1- responding to oxidative stress.</p

Mitochondrial activity as determined by MitoTracker in HeLa and CHP-212.

<p> Mitochondrial activity was measured by accumulated MitoTracker green fluorescence in mitochondria by means of flow cytometer (FL1-H channel). In all cases the bar represents the fluorescence emission of the dye; y-axis corresponds to the quantity of cells and data is representative of three independent experiments. Panel A corresponds to untreated control cells, Panel B to Cas/HeLa and Panel C to Cas/CHP-212.</p

AmplexRed peroxide determination by flow cytometry in HeLa and CHP-212.

<p>The quantity of cells expressing Hydrogen peroxide was detected with AmplexRed by flow cytometry in the FL2-H channel, whereas SSCH corresponds to side scatter channel. Panel A: untreated HeLa cells, Panel B: Cas/HeLa, Panel C: untreated CHP-212 cells, panel D: Cas/CHP-212. Positive controls corresponds to 3 h UV-irradiated cells. Data is representative of three independent experiments for each experimental condition.</p

FADD signaling in the caspase pathway is affected in HeLa cells treated with Cas II-gly.

<p>Image based in an analysis of the gene expression matrix in the Ingenuity database for Systems Pathways Analysis (IPA) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0054664#pone.0054664-Ingenuity1" target="_blank">[28]</a>. Molecules in green are transcriptionally down-regulated whereas molecules in red are transcriptionally over-expressed.</p

Top 20 GSEA - Cancer Modules for differentially expressed genes in HeLa cells treated with Cas II-Gly.

<p>Statistical significance was assessed by hypergeometric tests, corrected with the false discovery rate (FDR) algorithm.</p

Western Blot for molecules of interest in HeLa and CHP-212.

<p>Cells were previously treated with Cas II-gly at their corresponding IC, C = control cells without treatment, G = Cas II-gly treated cells. Western blot was done in the cytosolic portion after 6 h treatment for HeLa (panel A) and 2 h of treatment for CHP-212 (panel B), in order to detect active <i>caspase-3</i>, <i>caspase-8</i> cleaved, <i>cyt C</i>, <i>Bcl-2</i> and <i>Bax</i>. Protein expression was normalized to the -tubulin loading control. Each bar represents the mean SD (P0.05) from three independent experiments and expressed as Relative units measured by percentage of optical densitometry (% OD).</p

Structure of the Casiopeína II-gly molecule.

<p>Main figure depicts a full-atom quasi-3D rendering with optimized geometry. Grey atoms are Carbon, blue ones are Nitrogen, red ones are Oxygen, white are Hydrogen and the orange one is Copper as it can be seen at the inset.</p

HeatMap for hierarchical clustering of differential gene expression.

<p>HeLa Cells treated with Casiopeína II-gly vs untreated HeLa cells as Controls. We can notice the clear presence of groups of genes that are systematically, either up-regulated (red) or down-regulated (green) in treatment <i>versus</i> controls.</p

Glutathione determination by ELISA in HeLa and CHP-212.

<p>GSH intracellular index was measured by accumulated MCB fluorescence in a microtiter plate reader at 380/460 nm. GSH in non-treated cells was considered as a normalizing top value (100%); whereas negative control cells were MCB plus Cell Lysis Buffer. Data represent means standard deviation of three wells and experiments were replicated once. Differences between groups were assessed (marked with an asterisk) by ANOVA analysis with significance at P0.05.</p