Extended Data Fig. 1: The effects of LysoTag expression on lysosomes in vivo.
From: CLN3 is required for the clearance of glycerophosphodiesters from lysosomes
a, In the constitutive LysoTag mice, TMEM192–3×HA is expressed across all tissues examined as determined by immunoblotting using antibodies to the HA epitope. No difference in the expression of lysosomal markers LAMP1, Cathepsin B, C, and D is observed upon the expression of TMEM192–3×HA. Vinculin and AKT were used as loading controls. Numbers indicate molecular weights in kDa according to protein standards run on the same gel. Immunoblot is representative of two independent experiments. b, Normalized body weights of mice with the indicated genotypes at 5-9 weeks of age. Mice were weighed individually. The body mass of each mouse was normalized to the mean mass of sex-matched wild-type littermates before pooling by genotype for statistical analyses using a two-tailed unpaired t-test. (n = 25, 19, and 10 for Wild type, LysoTag+, and LysoTagLSL mice, respectively.). c,d, Measurement of lysosomal hydrolase activity in brain and liver tissues of the constitutive LysoTag mice shows no effect of TMEM192–3×HA expression on lysosomal function. For measuring Cathepsin B activity, its fluorogenic substrate was incubated with homogenates of the corresponding tissues (see methods). For determining β-hexosaminidase activity, fluorogenic 4-methylumbelliferyl (MUF)- N-acetyl-β-Glucosaminide was used as a substrate (see methods). Fluorescence was measured at 37 °C and data were shown following the subtraction of background fluorescence observed in the absence of homogenate (mean ± s.e.m.; n=4). e,f, Representative transmission electron micrographs showing lysosomes from brain and liver tissues of LysoTag- (control) and LysoTag+ mice. Lysosomes were identified by the presence of a single membrane and granular, electron-dense appearance. White and Red arrowheads indicate lysosomes in cells from LysoTag- and LysoTag+ mice, respectively. n = 2 mice per group and scale bar, 500 nm. The graph showing the mean diameter of lysosomes in multiple quantified cells. The diameter of lysosome in each group was generated by measuring lysosomes using ImageJ v1.52. (For brain, n = 42 and n = 55 measured lysosomes from LysoTag- and LysoTag+ mice, respectively. For liver, n = 51 and n = 56 measured lysosomes LysoTag- and LysoTag+ mice, respectively. Data presented as mean ± s.e.m. n.s: non-significant; Two-tailed unpaired t-test (e,f). g, Heatmap presentation of the enrichment of early- and late-endosomal markers as well as those for lysosome in the LysoIP performed from mouse liver tissue. Enrichment values are derived from Supplementary Table 1. For gel source data, see Supplementary Fig. 1.
