Figure 4.

Effect of rapamycin and everolimus on the mitochondrial integrity and autophagy in Treg cells were measured by flow cytometry analysis. Histograms (left) are representative single individual experiments of the corresponding bar-graphs (right) depicting the pooled (n=6) average mean fluorescence intensity (MFI)± SD from 5-day untreated (CTRL), 100nM rapamycin-treated (RAPA), or 100nM everolimus-treated (EVR) expanded Treg cells. (A) Mitochondrial mass (TOP) was measured with MitoTracker Green, which is independent from the mitochondrial membrane potential (mΔΨ). (BOTTOM) Changes in mΔΨ were analyzed by flow cytometry with the fluorescent probe TMRE. The resulting MFI within cells treated only with TMRE was corrected from the corresponding MFI of cells pre-treated with FCCP. Results for each condition are shown as the rate between TMRE increments (±FCCP) and the corresponding MitoTracker Green value, which normalize mΔΨ per equal mitochondrial mass. (B) The measurements of autophagic vacuole formation (TOP) were performed with the Cyto-ID autophagy detection kit. The accumulation of autophagosomes after the addition of the autophagic inhibitor Chloroquine (CLQ) for 6 hours before the addition of Cyto-ID Green solution was used to determine the autophagic flux (BOTTOM). The autophagosome flux is shown as the average MFI mean fluorescence intensity increase in cells pre-treated with CLQ compared to the cells in the absence of CLQ. Statistical differences among treatments were tested by Kruskal–Wallis ANOVA followed by post-hoc Wilcoxon test to assess treatment-specific differences. *p<0.05 and **p<0. 001 indicate significant pairwise differences; NS, no significant differences.