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. 2018 Jul 25;38(30):6682–6699. doi: 10.1523/JNEUROSCI.0054-18.2018

Figure 1.

Figure 1.

Aspirin induces lysosomal biogenesis in mouse primary astrocytes. A, Mouse primary astrocytes from WT C57BL/6 pups were treated with different concentrations of aspirin (2, 5, and 10 μm) under serum-free conditions for 24 h, followed by staining with LysoTracker Red to selectively label the lysosomes and acidic organelles in live cells. DAPI was used to stain the nuclei. Scale bar 10 μm. B, Quantification of the number of LysoTracker-positive puncta per cell. Data are shown as mean ± SEM for fold change with respect to the untreated control for at least 15 images per group from three independent set of experiments quantified using ImgaeJ. C, Astrocytes treated with 5 μm aspirin were subjected to TEM to monitor authophagic and lysosomal vesicles. Scale bar, 1 μm. D, Quantitative analysis of autophagic vesicle per cell represented as mean ± SEM; 20 images per condition were quantified using ImageJ. E–G, Mouse primary astrocytes treated with different doses (2, 5, and 10 μm) of aspirin followed by mRNA expression analysis at 8 h for the lysosomal genes Lanp2, Limp2, Npc1, and Cln2 by quantitative RT-PCR (E); immunoblot analysis at 24 h for LAMP2 TPP1 (F); and densitometric analysis of LAMP2 TPP1 protein expression (G). Data are shown as fold change (mean ± SD) with respect to untreated controls. H–J, Astrocytes treated with 5 μm aspirin for different time points (2, 6, 12, and 24 h), followed by determining mRNA expression for Lamp2 by quantitative RT-PCR analysis (H) and protein levels for LAMP2 TPP1 by Western blot analysis (I). Graph represents fold change (mean ± SD) relative to untreated controls. K, Immunofluorescence analysis of astrocytes treated with 5 μm aspirin for 24 h, followed by double labeling with LAMP2 and the astrocytic marker GFAP. Scale bar, 20 μm. L, TPP1 enzyme activity in astrocytes treated with different doses of aspirin for 24 h. Data are shown as mean ± SEM for the RFU fold change with respect to control. M, N, Cathepsin B and Cathepsin D enzyme activity in primary astrocytes treated with 2, 5, and 10 μm aspirin for 24 h. Data are shown as mean ± SD for the RFU fold change with respect to untreated controls. O, Immunocytochemistry analyzing Cathepsin D levels in astrocytes treated with 5 μm aspirin for 24 h. All statistical analysis were performed using Student's t test. *p < 0.05, **p < 0.001.