Fig. 3.
Increased granule-containing lysosomes (GCLs) and decreased macroautophagy in diabetic mice. a Immunofluorescence (IF) of insulin + proinsulin ((pro)insulin) (green) and CD63 (red) in β cells of 8-week-old BTBR +/+ and BTBR ob/ob mice. Arrowheads point to (pro)insulin/CD63 co-localization. Scale bar, 20 μm. b Electron microscopy of β cells in pancreatic islets isolated from 6-week-old BTBR +/+ and BTBR ob/ob mice. Yellow arrowheads point to granule-containing lysosomes (GCLs). Scale bar, 5 μm; for inset: 1 μm. Quantification of GCLs per cell view. N+/+ = 25 cells and Nob/ob = 36 cells from three mice per each group. ***P < 0.001, Mann–Whitney U-test. c Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) of GCLs of 6-week-old BTBR ob/ob mice. Sequential Z-slices showing GCL (yellow) and neighboring insulin granules (green). Scale bar, 0.2 μm. d 3D reconstruction of the GCL revealed by FIB-SEM in c. Detailed 3D reconstruction is shown in Supplementary Movie 3. e IF of (pro)insulin (green) and p62 (red) in β cells of 8-week-old BTBR +/+ and BTBR ob/ob mice. Scale bar, 20 μm. Quantification of p62 signal area: area of p62 signal (pixel²) over a fixed signal intensity threshold. N+/+ = 15; Nob/ob = 16, from three mice per each group. **P < 0.01, Mann-Whitney U-test. f Immunoblot of p62 using lysates of islets isolated from 8-week-old BTBR +/+ and BTBR ob/ob mice. GAPDH was used as a loading control. In b and e data are shown as mean ± SEM, source data are provided as a Source Data file. (a, e) Nuclei were stained with DAPI