OHCs lacking mucolipin 3 and 1 exhibit lysosomal membrane permeabilization. A–D, Immunoreactivity of galectin 3, a membrane-impermeable cytosolic lectin with high affinity for the luminal lysosomal glycocalyx (Aits et al., 2015), and LAMP1 reveal that lysosomal membranes in DKO OHCs are permeabilized. Galectin 3 is cytosolic in WT (A), ML1KO (B), and ML3KO (C) OHCs, and its expression level varied. D, In contrast, galectin 3 immunoreactivity has a vesicular pattern that is associated with LAMP1 signal in DKO OHCs. E, Super-resolution–structured illumination microscopy shows that galectin 3 is localized to lysosomal lumen. F–I, Immunoreactivity of cathepsin D and LAMP1 reveals that, upon lysosomal permeabilization, DKO lysosomes contain very low levels of cathepsin D. Cathepsin D has a vesicular expression pattern strongly associated with lysosomes in WT (F), ML1KO (G), and ML3KO (H) OHCs. I, In contrast, cathepsin D levels are low or undetectable in DKO lysosomes. Samples were from adult (∼P120) animals. Scale bars: A–D, F–I, 5 μm; E, 2 μm.