Impairment of lysosomal integrity by the loss of SPNS1.
(A) Immunofluorescence analysis. Parental, SPNS1-/-HeLa cells and SPNS1-/- HeLa cells expressing SPNS1-FLAG were immunostained with the LAMP-1 antibody. The left scatter plot shows the results of quantitative analysis of the size of LAMP-1-positive structures per cell in parental (WT) (n = 34401), SPNS1-/- HeLa (n = 40011), and SPNS1-/-HeLa expressing SPNS1-FL (n = 34812). The right scatter plot shows the number of LAMP-1-positive structures per area (μm2) in WT (n = 663), SPNS1-/- HeLa (n = 740), and SPNS1-/- HeLa expressing SPNS1-FL (n = 463). Blue indicates nuclei stained with Hoechst 33342. Each dot corresponds to individual data points, and the horizontal line indicates the mean. Statistical analysis was performed using the Šidák’s test after one-way ANOVA. Scale bars, 20 μm (main panels), 2 μm (inset panels). (B) Electron micrographs of cells indicated in (A). Boxed regions are enlarged and shown below. The number of lysosomes per area (μm2) of WT (n = 11), SPNS1-/- HeLa (n = 11), and SPNS1-/-HeLa expressing SPNS1-FL (n = 11) were quantified using the Image J software and presented in scatter plots. Each dot corresponds to individual data points, and the horizontal line indicates the mean. Statistical analysis was performed using the Šidák’s test after one-way ANOVA. Arrowheads indicate lysosomes. Bars, top panels: 5 μm, bottom panels: 500 nm. (C) Immunofluorescence microscopy. Liver sections from Spns1flox/flox and Spns1flox/flox;albumin-Cre mice at 3 months were stained with SPNS1 (green) and LAMP-1 (red) antibodies, and nuclei was stained with Hoechst 33342 (blue) as indicated. Boxed regions are enlarged and shown in the insets. Bar, 20 μm. (D) Electron micrographs of mouse liver sections, as indicated. Boxed regions are enlarged and shown on the right. Arrows indicate lysosome-like structures with undigested materials. Bars, 10 μm (left) and 1 μm (right). (E) Immunoblot analysis. Homogenates and lysosomal fractions prepared from the liver of Spns1flox/flox (n = 5) and Spns1flox/flox;albumin-Cre mice (n = 3) at 6 weeks of age were subjected to SDS-PAGE followed by immunoblotting with Cathepsin B and Actin antibodies. Scatter plots show the results of densitometric analysis of the intensity of the Cathepsin B band relative to that of the Actin band. Each dot corresponds to individual data points, and the horizontal line represents the mean. Statistical analysis was performed using the Welch’s t-test. (F) Cathepsin activity. The activity of Cathepsin B + L and that of Cathepsin B in lysosomal fractions shown in (E) was measured. Each dot corresponds to individual data points, and the horizontal line represents the mean. Statistical analysis was performed using the Welch’s t-test.