Fig. 6

Lysosomal abnormality in PGRN-deficient macrophages. a Representative images of LysoTracker Red DND-99 staining (red) and phase contrast images of RAW264.7 cells after transfection by siControl or siGrn. Scale bar: 10 μm. b Quantitative analysis of the fluorescence intensity of LysoTracker⁺ cells. Data are the means ± SEM (n = 6). ^*P <0.05 vs. siControl (Welch’s t-test). c Representative images of western blots showing immunoreactivity against LAMP1, cathepsin D, and β-actin. d–f Quantitative analysis of expression level of LAMP1 (d), pre-cathepsin D (e), and mature-cathepsin D (f) in RAW264.7 cells after transfection of siControl or siGrn and exposure to hypoxia (1% O₂, 12 h). Data are the means ± SEMs (n = 4). ^*P <0.05; ^**P <0.01 vs. siControl; ^#P <0.05; ^##P <0.01 vs. siControl + Hypoxia (one-way ANOVA followed by Tukey’s tests). g, h immunohistochemistry on the laser-injured eye of Grn^+/+ and Grn^−/− mice 7 days after the photocoagulation with anti-LAMP1 (white) and anti-cathepsin D (magenta) antibodies. Nuclei were stained with Hoechst 33342 (blue) and the CNV regions were visualized by FITC-dextran (green). Yellow arrows show LAMP1⁺ or cathepsin D⁺ cells infiltrated into the CNV area. Scale bar: 100 μm. i, j Level of expression of sortilin in RAW264.7 cells after transfection by siControl or siGrn and exposure to hypoxia. Data are the means ± SEMs (n = 4). ^*P <0.05; ^**P <0.01 vs. siControl (one-way ANOVA followed by Tukey’s tests)