Fig. 4: Expression of ER-targeted Nb4, Nb9, and Nb16, and of lysosome-targeted Nb16, increased lysosomal GCase activity in live cells.
From: Developing nanobodies as allosteric molecular chaperones of glucocerebrosidase function
A Schematic representation of the lysosomal GCase activity assay, performed using the PFB-FDGlu substrate in HEK293 live cells expressing different Nbs. Created in BioRender. Plotegher, N. (2025) https://BioRender.com/efdob53. B Expression of ER-Nb4, ER-Nb9, and ER-Nb16 significantly increased the lysosomal GCase activity by about 15%, as compared to ER-Mock transfected cells (n = 12 for ER-Mock and ER-Nb9 in 6 independent experiments, n = 6 for ER-Nb1, ER-Nb4 and ER-Nb16 in 3 independent experiments, data represented as violin plots, Shapiro–Wilk test for normality, Ordinary One Way Anova with multiple comparisons, DF Nbs = 4, DF residual = 31, F value = 9.58). C Only lysosomal-Nb16 improved GCase enzymatic activity (n = 15 for lyso-Mock, n = 9 for lyso-Nb9, n = 6 for ER-Nb1 and ER-Nb4 in 3 independent experiments, data represented as violin plots, Shapiro–Wilk test for normality, Ordinary One Way Anova with multiple comparisons, DF Nbs = 4, DF residual = 37, F value = 18.2). D Schematic representation of the DQ-BSA assay performed to evaluate lysosomal proteolytic activity upon Nb overexpression in HEK293T cells. Created in BioRender. Plotegher, N. (2025) https://BioRender.com/b8bx884. E No changes in lysosomal proteolytic activity in HEK293T cells overexpressing the selected ER-Nbs (n = 8 for ER-Mock, n = 9 for ER-Nb1, ER-Nb4, ER-Nb9, ER-Nb16 in three independent experiment, data represented as violin plots, Shapiro–Wilk test for normality, Ordinary One Way Anova with multiple comparisons, DF Nbs = 4, DF residual = 39, F value = 1.716). F No changes in lysosomal proteolytic activity in HEK293T cells overexpressing the selected lysosmal-Nbs (n = 9 for lyso-Mock, n = 10 for lyso-Nb1, n = 6 for lyso-Nb4, n = 7 for lyso-Nb9, n = 8 for lyso-Nb16 in four independent experiments, data represented as violin plots, Shapiro–Wilk test for normality, Ordinary One Way Anova with multiple comparisons, DF Nbs = 4, DF residual = 35, F value = 0.2793). Source data are deposited as Source Data files on Zenodo. GCase glucocerebrosidase, Nb nanobody, PFB-FDGlu 5-(Pentafluorobenzoylamino)Fluorescein Di-β-D-Glucopyranoside.
