Fig. 3: Effect of everolimus on high glucose-induced cell death compared with rapamycin.

A UCB-MSCs were treated with D-glucose (25 mM) for various times (0–72 h). Protein expression levels of mTOR, p-mTOR (Ser 2448), S6K1, and p-S6K1 (Thr 389) were determined by western blot analysis (n = 4). B UCB-MSCs were pretreated with 100 nM everolimus or rapamycin for 30 min, followed by D-glucose (25 mM) treatment for 72 h. Intracellular glucose influx in UCB-MSCs was measured by a glucose uptake assay (n = 4). C UCB-MSCs were pretreated with various concentrations (0.1–1 μM) of everolimus or rapamycin for 30 min followed by D-glucose (25 mM) treatment for 72 h. mtROS levels were assessed by MitoSOX staining (n = 10). D, E UCB-MSCs were pretreated with everolimus (100 nM) for 30 min followed by D-glucose (25 mM) treatment for 72 h. D Mitochondrial superoxide generation was visualized by live-cell staining. UCB-MSCs were stained with MitoSOX (red), MitoTracker (green), and Hoechst 33342 (blue) (n = 10). Magnification × 1000. Scale bars are 25 μm. E Mitochondrial membrane potential was assessed by TMRE staining (n = 10). F,G UCB-MSCs were pretreated with various concentrations (0.1–1 μM) of everolimus or rapamycin for 30 min followed by D-glucose (25 mM) treatment for 72 h. F Cell viability was measured by the trypan blue exclusion assay (n = 9). G UCB-MSCs were pretreated with various concentrations (0.1–1 μM) of everolimus or rapamycin for 30 min followed by D-glucose (25 mM) treatment for 72 h. Cytotoxicity in UCB-MSC-conditioned medium was measured using an LDH release detection kit (n = 7). H UCB-MSCs were pretreated with everolimus (100 nM) for 30 min followed by D-glucose (25 mM) treatment for 72 h. Protein expression levels of Bax, Bcl-2, and cleaved caspase-9 were determined by western blot analysis, (n = 4). All quantitative data are presented as the mean ± standard deviation from independent experiments. N.S., no significance, *p < 0.05.