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 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

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