Figure 4

Rapamycin enhances endocytosis of Dil-oxLDL in mesenchymal stem cells and targets them to autophagosomes. (a) BM-MSCs plated in 96 wells were either tested naive or activated with 1 µM of rapamycin for 3 hr before treatment with Dil-oxLDL (100 µg/mL). Cells were washed with warm medium at the indicated times and imaged or read for fluorescence uptake at 530/590 nm (Ascent fluorometer). AFUs were plotted against time (*p < 0.01 vs. rapamycin alone; 2 similar experiments, t test). (b) Deconvolution images of Dil-oxLDL (red) within MSCs shown before and after rapamycin activation. (c) MSCs were activated with rapamycin (1 µM) and added with Dil-oxLDL after 90 min, washed and incubated with freshly added rapamycin. At 4, 24, 48 and 72 hr, the cells were stained using cyto-ID for autophagosomes and imaged using deconvolution microscopy. Representative images from 50 cells (triplicates) analyzed from three similar experiments are shown. Naïve MSCs containing Dil-oxLDL and autophagic puncta are shown in Supplemental Fig. S4. Rapamycin induced Dil-oxLDL (red) to colocalize with autophagosomes (inset). Nuclei are stained blue using DAPI (bar = 5 µM). (d) Quantitation of colocalization of Dil-oxLDL with autophagic puncta is shown.