Figure 11

Digitonin changes membrane biophysical properties in a Chol-dependent manner. (A–D) LUVs composed of eSM:ePC (1:1, striped bars or dashed lines), ePC:Chol (1:1, grey bars or lines) or eSM:ePC:Chol (1:1:1, black bars or lines) were incubated with 1% DMSO or increasing digitonin/lipid ratios as indicated at 25 °C. (A) LUV size was determined by dynamic light scattering. Unpaired t-test was used to compare control versus digitonin-treated LUVs. (B) Fluidity was determined by GPex values on LUVs labeled with Laurdan. One-way ANOVA with Dunnett’s post tests to compare control versus digitonin-treated LUVs. *p < 0.05; **p < 0.01; ***p < 0.001. (C,D) Membrane fusion and permeability were assessed by R₁₈ fluorescence dequenching (C) and calcein self-quenching release (D), respectively. All the results are means of two independent experiments in triplicates. (E) GUVs composed of eSM:ePC, ePC:Chol or eSM:ePC:Chol were labeled with Rhod-PE (red) and NBD-PE (green) and were incubated with a digitonin/lipid ratio of 20 at room temperature. After 2 min following digitonin treatment, pictures were taken with a Zeiss wide-field fluorescence microscope (Observer.Z1) using a plan-Apochromat 40x/1.4 oil Ph3 objective. Scale bars, 20 μm. Images are representative of two independent experiments.