Fig. 5: MDV-lysosomal degradation of CiC in aged MSCs.

a,b, Representative images after immunostaining against CiC (a) and quantification of MFI of young and aged control cells, following indicating treatments (b). Nuclei were stained with DAPI. Scale bar, 10 μm. n = 3 biologically independent replicates and 1 representative replicate (n = 27 cells for young, n = 25 cells for old, n = 20 cells for E64d and n = 21 cells for BafA1) is shown in b. c, Representative images after immunostaining of cells from a against CiC and LAMP2 (right) and intensity plots of line scans showing the intensity of CiC (green line) and LAMP2 (red line). Shaded regions highlight the CiC signal in areas with increased lysosomal intensity. Nuclei were stained with DAPI. Scale bar, 10 μm. n = 3 biologically independent replicates. d,e, Representative images after immunostaining against CiC (d) and quantification of MFI of young control and young E64d-treated cells (e). Nuclei were stained with DAPI. Scale bar, 50 μm. n = 3 biologically independent replicates and merged results (n = 68 cells for young_control and n = 80 cells for young_BTA samples) are shown. f, Representative images after immunostaining of cells from d against CiC and LAMP2 (right) and intensity plots of line scans, as described in c. Nuclei were stained with DAPI. Scale bar, 50 μm. n = 3 biologically independent samples. g, Representative images of MDVs highlighted with arrows, in young and aged cells and higher magnification of the areas in the boxes. Scale bars, 500 nm. n = 3 biologically independent samples. h,i, Representative images after immunostaining against TOMM20, CiC and PDH and quantification of TOMM20+/PDH−/CiC+ MDVs (h), shown in higher magnification of areas in the box. n = 2 biologically independent replicates and results from 1 representative replicate (n = 18 cells for young and n = 23 cells for old samples) is shown as violin plot (i). Nuclei were stained with DAPI. Scale bar, 8 μm. Distribution of data points in b, e and i is shown as violin plots, where the mean is indicated by a solid line and the quartiles are indicated with dashed lines. Statistical significance in b, e and i was determined using two-sided unpaired t-test.