Loss of Cbs disrupts lysosomal homeostasis in mouse liver and kidney. (A) Representative images of liver sections stained with antibodies of LAMP1 (red) and EEA1 (green). Boxed regions are magnified in merged images. Lysosomes and endosomes are indicated with yellow and purple arrows, respectively. Bars, 5 μm. (B) Left: Representative images of LysoTracker Red staining in hepatocytes. Original staining images and the same images indicated with fluorescence intensity are shown Boxed regions are magnified in “Zoom” image. Bars, 5 μm. Quantifications of lysosomal area and LysoTracker Red intensity (A.U., arbitrary units) are shown in the middle and right panels, respectively. 30 lysosomes in 10 hepatocytes from 3 mice were analyzed for each genotype. (C) Western blotting of CTSB (left) and CTSD (right) in liver lysates of WT and Cbs^−/− mice. GAPDH was used as the loading control. Fold changes of matured CTSB (mature CTSB/total CTSB/GAPDH) and matured CTSD (mature CTSD/total CTSD/GAPDH) are shown at the bottom. (D) Left: Immunostaining of LAMP1 and LC3 in hepatocytes isolated from WT and Cbs^−/−mice. Boxed regions in each row are magnified and shown in the merge images. Bars, 5 μm. Middle and right: Quantifications of LC3 puncta (middle, n = 10 hepatocytes) and colocalization of LC3 with LAMP1 (right, n = 30 lysosomes in 10 hepatocytes). (E) TEM images of hepatocytes (top left) and macrophages (bottom left) from WT and Cbs^−/− mouse livers. Yellow arrowheads: normal lysosomes; orange arrowheads: damaged lysosomes; magenta arrowheads: enlarged cargo-filled lysosomes. Scale bars, 1 μm. Right panels show quantification of lysosome diameters in hepatocytes (top) and macrophages(bottom left) (≥50 lysosomes analyzed from 2 WT and 3 Cbs^−/− mice); percentage of damaged lysosomes in macrophages (bottom right) (≥20 macrophages analyzed from 2 WT and 3 Cbs^−/− mice). Scale bars, 1 μm. (F) TEM images of kidney sections from WT and Cbs^−/− mice with same lysosome labeling scheme as in A. Quantification of lysosomal parameters shown on right. Scale bars, 1 μm. Quantification of lysosomal diameter (≥50 lysosomes analyzed from 2 mice per genotype) and percentage of damaged lysosomes (≥10 cells analyzed from 2 mice per genotype) in kidney sections. For all quantifications, data are presented as mean ± SEM. Statistical significance was calculated using the two-sided Student’s t test. ****P < 0.0001. Source data are available for this figure: SourceData F8.