Immunoblot evaluation of cleaved PARP and caspase 3.

<p>RKO cells treated with vehicle, 1.5, or 5 µM JS-K for 24 hrs. Protein was separated by SDS-PAGE and detected with immunoblot analysis. A dose-dependent increase in cleaved PARP and caspase 3 (CASP3) was observed with more cleaved material in the TR1 knockdown. GAPDH was evaluated as a loading control.</p

Summary of Trx redox status following TR1 knockdown and JS-K treatment: Percent Trx in each redox state (% total).

<p>Summary of the 6 possible redox states of Trx in RKO cells with endogenous TR1 (si-Scramble) or with endogenous TR1 knocked-down (si-TR1) following treatment with 5 µM JS-K for 90 min. Trx does show a shift to more oxidized states in the cells treated with JS-K with TR1 knocked down.</p

MTT-based viability assays for RKO cells following increasing concentrations of redox modulators.

<p>Cells with endogenous TR1 (si-Scramble, filled square), and cells with TR1 knocked-down by siRNA (si-TR1, filled circle) were compared at equivalent doses of the redox modulators. A) RKO cells were treated with increasing concentrations of H₂O₂ and the viability was measured after a 24 hrs exposure. No significant differences were observed. B) RKO cells were treated with increasing concentrations of 4-HNE and the viability was measured after a 24 hrs exposure. The si-TR1 cells displayed ∼2-fold increased sensitivity to 4-HNE. C) RKO cells were treated with increasing concentrations of JS-K and the viability was measured after a 24 hrs exposure. The si-TR1 cells displayed ∼6-fold increased sensitivity to JS-K.</p

Evaluation of the 6 possible redox states of Trx.

<p>In cells without stimulation (left three lanes), Trx is primarily in the reduced state, even with TR1 knocked-down and the C-terminal mutant TR1 expressed. With 1 mM diamide stimulation for 30 min (right three lanes), cells with endogenous TR1 demonstrate more reduced Trx than cells with endogenous TR1 knocked-down with siRNA.</p

Characterization of TR1 levels and activity in RKO cells.

<p>Cells were exposed to siRNA directed against TR1 for a total of 96 hrs, and a Sec-deficient C-terminal mutant TR1 was induced for the last 24 hrs. A) Immunoblot analysis of TR1 protein expression following siRNA treatments and induction of the Sec-deficient mutant TR1. B) TR biochemical activity measured in cell lysates by monitoring NADPH oxidation in an assay that is dependent on Trx and uses insulin is the final electron acceptor. The first bar on the left represent the activity of the control (si-Scramble) without Trx added to the reaction mix, indicating background signal. C) Cell-based TR activity as measured by lipoic acid reduction in a colorimetric assay using Ellman's reagent. The first bar on the left represent the activity of the control (si-Scramble) without lipoic acid added to the reaction mix. The lysates from cells with TR1 knocked down show significant reductions in activity compared to the control (si-Scramble) in both assays (***, p<0.001).</p

Summary of Trx redox status following TR1 knockdown and Sec-deficient TR1 expression: Percent Trx in each redox state (% total).

<p>Summary of the 6 possible redox states, from the most oxidized (state 1) to the least (state 6) of Trx in RKO cells with endogenous TR1 (si-Scramble), with endogenous TR1 knocked-down (si-TR1), or endogenous TR1 knocked-down but with induced Sec-deficient mutated TR1 (si-TR1+mTR1). Since the majority of the Trx was found to be in the reduced state, we stimulated with 1 mM diamide for 30 min to oxidize the cells and those cells without endogenous TR1 display more oxidized Trx than cells with endogenous TR1.</p

Protease activity as a measure of viability, cytotoxity and effector caspase activity.

<p>RKO cells treated with 1.5 µM JS-K for 24 hrs. were assessed for A) viability, B) cytotoxicity, and C) caspase-3/7 activity. In separate experiments, cells were incubated with Z-Asp-CH₂-DCB, a broad spectrum, competitive caspase inhibitor, to determine that the caspase assay was indeed demonstrating effector caspase activity (C). RKO cells with TR1 knocked-down demonstrate significantly greater losses in viability, increased cytotoxicity, and increased caspase-3/7 activity following JS-K treatment than RKO cells with endogenous TR1 (**, p<0.01; ***, p<0.001; †††, p<0.001).</p

RKO cellular redox state following JS-K treatment as measured by glutathione redox status.

<p>GSH measurements were made following treatment with 5 µM JS-K for 24 hrs., and the ratio of the reduced GSH to the total GSH was measured. No significant differences were observed among the treatment groups.</p

Cell growth kinetics of RKO cells with modulated TR1 levels.

<p>A scrambled siRNA was used as a control to measure the basal growth rate (filled square) TR1 was knocked-down by siRNA (filled circle) and with induction of the Sec-deficient, C-terminal mutant TR1 (filled triangle). No significant differences in growth rates were observed as the solid lines used to calculate the growth rates for these conditions are nearly parallel.</p

Transcriptional Responses of Human Aortic Endothelial Cells to Nanoconstructs Used in Biomedical Applications

Understanding the potential toxicities of manufactured nanoconstructs used for drug delivery and biomedical applications may help improve their safety. We sought to determine if surface-modified silica nanoparticles and poly­(amido amine) dendrimers elicit genotoxic responses on vascular endothelial cells. The nanoconstructs utilized in this study had a distinct geometry (spheres vs worms) and surface charge, which were used to evaluate the contributions of these parameters to any potential adverse effects of these materials. Time-dependent cytotoxicity was found for surfaced-functionalized but geometrically distinct silica materials, while amine-terminated dendrimers displayed time-independent cytotoxicity and carboxylated dendrimers were nontoxic in our assays. Transcriptomic evaluation of human aortic endothelial cell (HAEC) responses indicated time-dependent gene induction following silica exposure, consisting of cell cycle gene repression and pro-inflammatory gene induction. However, the dendrimers did not induce genomic toxicity, despite displaying general cytotoxicity