Figure 4. Deletion of ERp57 blunted the therapeutic effects of ND7 and PGRN in GD patient fibroblasts.

(A) Diagram of the CRISPR/Cas9 technique for construction of human ERp57 KO GD type 2 fibroblasts L444P. (B) The levels of ERp57 in ERp57 KO L444P and control L444P fibroblasts, as-sayed by Western Blotting. (C) The lysosomal content in L444P with or without ND7 and PGRN treatment. L444P fibroblasts were stimulated with lipid lysis and treated with recombinant ND7 (5 μg/mL) or PGRN (10 μg/mL) for 24 hours. PBS used as control. The cells were stained with LysoTracker Red. The fluorescence intensity of the LysoTracker Red was determined by the live fluorescence microscopy imaging. (D) Quantification of mean fluorescence intensity of C analyzed using Image J. (E) The accumulation of β-glucosylceramide (β-GlcCer, green) in L444P fibroblasts with or without PGRN or ND7 treatment was analyzed by immunofluorescence staining with antibody against β-GlcCer. (F) Quantification of mean fluorescence intensity of E. (G) The lysosomal content in control or ERp57 KO L444P with or without ND7 and PGRN treatment. L444P fibroblasts were stimulated with lipid lysis and treated with recombinant ND7 (5 μg/mL) or PGRN (10 μg/mL) for 24 hours. PBS used as control. The cells were stained with LysoTracker Red. The fluorescence intensity of the LysoTracker Red was determined using SpectraMax i3x plate reader at excitation/emission of 647/668 nm. (H) The GCase activity in control or ERp57 KO L444P fibroblasts with or without ND7 and PGRN treatment were assayed by released 4MU-Glc. Data are shown as mean ± SD of 3 independent experiments. One-way ANOVA tests. *, p<0.05, **, p<0.01, ***p<0.001. Scale bar=100 μm.