Fig 1: Opposite effect of CBE and PPT1 deficiency on lysosomal acidification. (A) Representative images of primary cortical cultured neurons treated with CBE for 7 days at different concentrations and stained with anti-MAP2 antibody and LysoTracker Red DND-99. Scale bar = 50 µm. (B) Graphs show mean ± SEM LysoTracker-red-DND-99-integrated fluorescence intensity and MAP2-positive area from one representative experiment. n = 36–54 images. (C) Representative agarose gel showing confirmation of Ppt1 DNA editing by T7EI digestion in cultured neurons 2 weeks after AAV2/1 infection. Cortical cultured neurons were infected with AAV2/1 expressing two single-guide Ppt1 RNA sequences (sg) at 3 DIV. (D) Ppt1 mRNA expression in cultured neurons 2 weeks after AAV2/1 infection. n = 3 independent experiments. (E) Representative immunoblots with anti-ATP6V1A and ß-actin using cortical cultured neuronal lysates 2 weeks after AAV2/1 infection. Full blots for this other panels are shown in Supplementary Fig. 11. (F) Graph shows mean ± SEM of the immunoblot signal from E. n = 3 independent experiments. (G) Representative immunoblots with anti-ATP6AP1, ATP6V1A and ß-actin using cortical lysates from WT and Ppt1-/- mice. (H) Graphs show mean ± SEM of the immunoblot signal from G. n = 6–9 mice per group. (I) Representative images of primary cortical cultured neurons infected with AAV2/1. Two weeks later, neurons were stained with anti-MAP2 antibody and LysoTracker Red DND-99. Scale bar = 100 µm. (J) Graphs show mean ± SEM LysoTracker-red-DND-99-integrated fluorescence intensity and MAP2-positive area from one representative experiment. n = 50–58 images. (K) Representative immunoblots with anti-ATP6AP1, ATP6V1A and ATPv0a1 and ß-actin using cerebral cortex lysates from 5-month-old WT, Ppt1-/-, Tmem106b-/- and Ppt1-/-; Tmem106b-/- mice. (L) Graphs show mean ± SEM of the immunoblot signal from K. n = 6–7 mice per group