Fig. 2

ROCK1 decreases lysosomal biogenesis and impairs lysosomal acid environment. a Schematic illustration of the experimental procedure. Image created with BioRender.com. b Heatmap of mRNA expression of ROCK1 and lysosome-related markers detected with qRT-PCR in HEK-293T cells transfected with control or ROCK1-overexpression plasmid for 48 h. n = 3. Red color, higher expression; blue color, lower expression. c Immunoblotting of lysosome-related markers in HEK-293T cells and quantitation of their protein levels. n = 3. d Transmission electron microscopy of lysosomes in HEK-293T cells. The number of lysosomes was analyzed. n = 10 cells per group. Scale bar, 2 μm. White arrows indicate lysosomes. e Lysotracker Red, Magic Red B and DQ BSA staining in HEK-293T cells. Fluorescence intensities were analyzed. n = 5. Scale bars, 25 μm. f Standard curve of LysoSensor to determine the lysosomal pH values of HEK-293T cells. g Lysosomal pH of HEK-293T cells was determined by LysoSensor. n = 5. h Schematic illustration of the experimental procedure. Image created with BioRender.com. i AAV-Vector and AAV-ROCK1 were successfully microinjected into the hippocampus of WT mice, respectively. GFP (green) was used to visualize viral diffusion. Scale bar, 200 μm. j qRT-PCR analysis of expression levels of indicated mRNAs. n = 3. k Transmission electron microscopy of lysosomes in the hippocampus. The number of lysosomes was analyzed. n = 9 slices from 3 mice. Scale bars, 1 μm. *P < 0.05, **P < 0.01, ***P < 0.001 versus vector (b-e, g) or AAV-Vector group (j, k), Student’s t-test