The Effect of PAO (1.0 µM) on Glutathione Concentration.

<p>The Effect of PAO (1.0 µM) on Glutathione Concentration.</p

The relationship between the amount of S-glutathionylation and the vicinal dithiols-containing molecules.

<p>HEK 293 cells were pretreated with 1 µM PAO for 30 minutes before treating with 0.25 mM diamide or 0.5 mM hydrogen peroxide. The preparation of PAO was described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004015#s3" target="_blank">Methods</a>, and the effect of PAO on cellular glutathione was shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004015#pone-0004015-t003" target="_blank">Table 3</a>. Cell lysates were prepared, and modified proteins were detected as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0004015#s3" target="_blank">Methods</a>.</p

Protein S-glutathionylation in HEK 293 cells.

<p>(A) The pattern of overall S-glutathionylated proteins is shown in a time course experiment with 0.5 mM diamide or 0.5 mM hydrogen peroxide-treated HEK 293 cells. Equal amount of cell lysates were loaded and separated by a 12% SDS gel under non-reducing condition. S-glutathionylated proteins were detected by Western blot using anti-glutathione monoclonal antibody. Some predominately modified proteins are indicated by arrows. (B) The same amount of lysates from 1(A) were separated in a SDS-gel under reducing condition (50 mM DTT) and the membrane was blotted and stained the same way as in 1(A). The loss of signals indicated the anti-glutathione monoclonal antibody was selectively detecting glutathione moiety on proteins.</p

The Effect of Diamide (0.5 mM) and Hydrogen Peroxide (0.5 mM) on Glutathione Concentration.

*<p>Total GSH is calculated by [GSH]+2[GSSG].</p

A Molecular Umbrella Approach to the Intracellular Delivery of Small Interfering RNA

A series of diwalled and tetrawalled molecular umbrellas have been synthesized using cholic acid, spermidine, and lysine as starting materials. Coupling of these molecular umbrellas to an octaarginine peptide afforded agents that were capable of promoting the transport of small interfering RNA to HeLa cells, as judged by the knockdown of enhanced green fluorescent protein expression. The efficiency of this knockdown was found to increase with an increasing number of facially amphiphilic walls present, and also when a cleavable disulfide linker was replaced with a noncleavable, maleimido moiety; i.e., a group that is not susceptible to thiolate-disulfide interchange. The knockdown efficiency that was observed for one tetrawalled molecular umbrella–octaargine conjugate was comparable to that observed with a commercially available transfection agent, Lipofectamine 2000, but the conjugate showed less cytotoxicity