P4 induces the aberrant lysosomal function.

(A) PK-15 cells were treated with P4 (10 μM) for 8 hours. Co-localization analysis of intracellular cholesterol (filipin) with mitochondria, peroxisomes, endoplasmic reticulum (ER), and lysosomes was performed by immunofluorescence and filipin staining. Scale bar: 10 μm. (B) PK-15 cells were treated with vehicle or P4 (10 μM). Lysosomal pH was measured 8 hours post-treatment. (C) PK-15 cells were transfected with a vector or NPC1-GFP plasmid for 24 hours. Afterward, cells were treated with vehicle or P4 (10 μM) for an additional 8 hours. Co-localization analysis of LAMP1 (red) with NPC1-GFP was performed by immunofluorescence analysis. Scale bar: 10 μm. (D) Quantification of the perinuclear index of LAMP1 in PK-15 cells from (C). (E) PK-15 cells were treated with P4 (10 μM) for 0–8 hours. Co-localization analysis of fluo-3 with LAMP1 was performed by immunofluorescence analysis. Scale bar: 10 μm. (F) Pearson’s correlation coefficients of fluo-3 with LAMP1 in PK-15 cells from (E). Data are expressed as the mean ± SD of 3 independent experiments. P-values were determined by Student’s t-test. P (TIF)