Cell blebs and cytoskeleton under different DMSO concentrations.

<p><b>(A)</b> The bleb and cytoskeleton were observed by an inverted fluorescence microscope (membrane: red; cytoskeleton: green). <b>(B)</b> The bleb and cytoskeleton were observed by a confocal microscope (cytoskeleton: green; nucleus: blue). <b>(C)</b> The fluid flows in the formation of blebs under a hypoosmotic condition (0.1×PBS) and a hyperosmotic condition (25% DMSO in PBS). The experiments were repeated 3 times.</p

Stepwise addition of DMSO.

<p><b>(A)</b> Comparison of the mortality rate of cells between stepwise and single-step addition. <b>(B)</b> Bleb index in the stepwise addition method. HeLa cells were treated with 20% DMSO for 30 minutes, and the solution was removed quickly and changed to 40% DMSO for 30 minutes. It was then changed to 60% DMSO for 30 minutes and, finally, to 80% DMSO. The inverted fluorescence microscope was used to observe dead cells labeled by PI and Hoechst. For <b>(A)</b>, the number of cells used was approximately 500 and the experiment was repeated 5 times. For <b>(B)</b>, the number of cells used was approximately 40. **p<0.01 was considered statistically significant.</p

Cell blebs induced by the addition of CPAs.

<p>Various concentrations of <b>(A)</b> DMSO and <b>(B)</b> glycerol were applied to HeLa cells for 30 minutes. The development of cell blebs during the first 3 minutes was observed as the initial state and after 30 minutes as the stable state. Initiate: 3 minutes, and Stabilized: 30 minutes. The experiments were repeated 3 times.</p

Life cycle of a dynamic bleb.

<p><b>(A)</b> the inflation and retraction of one bleb (black arrows); <b>(B)</b> the actin microfilament reorganization during the bleb inflation and retraction; <b>(C)</b> the comparison of the inflation and retraction time between DMSO and glycerol. For <b>(A)</b> and <b>(B)</b>, the experiments were repeated 3 times. For <b>(C)</b>, the number of cells used was approximately 20.</p

The autophagy induced by the addition of CPAs.

<p><b>(A)</b> GFP-LC3/HeLa cells were treated with various concentrations of DMSO, and GFP green fluorescence dots appeared in cells. <b>(B)</b> LC3 conversion was determined by western blot in HeLa cells treated with different concentrations of DMSO. <b>(C)</b> Effect of DMSO on the autophagy rate. <b>(D)</b> GFP-LC3 /HeLa cells were inhibited by 3-MA, and then stimulated by 30% DMSO. Shrinkage of cell nuclei is a hallmark of apoptosis. <b>(E)</b> Autophagy reduced the apoptosis in the presence of 30% DMSO. **p<0.01 was considered statistically significant. The experiments were repeated 5 times. The number of cells used was approximately 500.</p

Effect of the concentration of CPAs: (A) number of cell blebs; (B) total area of cell blebs; (C) bleb index; (D) mortality rate of cells; (E) schematic of A_lip and A_cyto.

<p>HeLa cells were treated with a series of solutions containing different amounts of DMSO or glycerol, as well as the fluorochromes Hoechst and PI. After 30 minutes, when cells were stable, an inverted fluorescence microscope was used to observe cell death. For <b>(A)</b>, <b>(B)</b> and <b>(C)</b>, the cell number was approximately 40. For <b>(D)</b>, the cell number was approximately 500 and the experiment was repeated 5 times. For <b>(E)</b>, the red boundary denotes the lipid bilayer and the green boundary denotes the cortical cytoskeleton.</p

Nanoparticle as Signaling Protein Mimic: Robust Structural and Functional Modulation of CaMKII upon Specific Binding to Fullerene C60 Nanocrystals

In a biological environment, nanoparticles encounter and interact with thousands of proteins, forming a protein corona on the surface of the nanoparticles, but these interactions are oftentimes perceived as nonspecific protein adsorption, with protein unfolding and deactivation as the most likely consequences. The potential of a nanoparticle–protein interaction to mimic a protein–protein interaction in a cellular signaling process, characterized by stringent binding specificity and robust functional modulation for the interacting protein, has not been adequately demonstrated. Here, we show that water-suspended fullerene C60 nanocrystals (nano-C60) interact with and modulate the function of the Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), a multimeric intracellular serine/threonine kinase central to Ca²⁺ signal transduction, in a fashion that rivals the well-documented interaction between the NMDA (<i>N</i>-methyl-d-aspartate) receptor subunit NR2B protein and CaMKII. The stable high-affinity binding of CaMKII to distinct sites on nano-C60, mediated by amino acid residues D246 and K250 within the catalytic domain of CaMKIIα, but not the nonspecific adsorption of CaMKII to diamond nanoparticles, leads to functional consequences reminiscent of the NR2B–CaMKII interaction, including generation of autonomous CaMKII activity after Ca²⁺ withdrawal, calmodulin trapping and CaMKII translocation to postsynaptic sites. Our results underscore the critical importance of specific interactions between nanoparticles and cellular signaling proteins, and the ability of nano-C60 to sustain the autonomous kinase activity of CaMKII may have significant implications for both the biosafety and the potential therapeutic applications of fullerene C60

Inhibition of autophagy enhances the anticancer activity of silver nanoparticles

<div><p>Silver nanoparticles (Ag NPs) are cytotoxic to cancer cells and possess excellent potential as an antitumor agent. A variety of nanoparticles have been shown to induce autophagy, a critical cellular degradation process, and the elevated autophagy in most of these situations promotes cell death. Whether Ag NPs can induce autophagy and how it might affect the anticancer activity of Ag NPs has not been reported. Here we show that Ag NPs induced autophagy in cancer cells by activating the PtdIns3K signaling pathway. The autophagy induced by Ag NPs was characterized by enhanced autophagosome formation, normal cargo degradation, and no disruption of lysosomal function. Consistent with these properties, the autophagy induced by Ag NPs promoted cell survival, as inhibition of autophagy by either chemical inhibitors or <i>ATG5</i> siRNA enhanced Ag NPs-elicited cancer cell killing. We further demonstrated that wortmannin, a widely used inhibitor of autophagy, significantly enhanced the antitumor effect of Ag NPs in the B16 mouse melanoma cell model. Our results revealed a novel biological activity of Ag NPs in inducing cytoprotective autophagy, and inhibition of autophagy may be a useful strategy for improving the efficacy of Ag NPs in anticancer therapy.</p></div