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. 2022 Mar 24;23(7):3536. doi: 10.3390/ijms23073536

Figure 2.

Figure 2

Acid β-glucosidase (β-Glu) enzyme activity, stability, and glycosylation in compound-treated patient fibroblasts. (a–c) β-Glu activity in ambroxol/isofagomine (ABX/IFG)-treated cells. Three cell lines of unrelated patients harboring either compound heterozygous p.Asn370Ser/84GG (GM00852, GM00372) mutations or wildtype GBA1 (GM01653) were cultured for 5 days in absence and presence of ABX (black columns) or IFG (grey columns). The cells were harvested following a 6 h washout period, lysed, and subjected to enzyme measurement using 3 mM 4-methylumbelliferyl-β-D-glucopyranoside (4-MUG). Data are provided as mean ± SEM from 6–12 (a,b) and 3 (c) independent experiments. Differences between the groups were analyzed using one-way ANOVA. Post-hoc Dunnett test was used to analyze each column with the respective value of the untreated cells (significance level is represented by *, **, *** as p-values of 0.05, 0.01 and 0.001); n.s., not significant. (d) Cycloheximide (CHX) chase experiment. Patient fibroblasts of line GM00852 and control cells (GM01653) were cultured for 5 days in the presence or absence of treatment. Then, in addition to treatment, CHX was added for at 0, 2, 4, 6, 8, 12, and 24 h to study intracellular stability of β-Glu. Cell lysates containing 20–30 μg were digested with PNGase F to obtain a singular protein band in immunoblot using anti-β-Glu (2E2) antibody. (e) Decay curves of β-Glu in fibroblast cells. Each β-Glu band was normalized to its corresponding β-Actin band (not shown). All normalized values obtained were referenced to the 0 h time point of each treatment series before CHX chase start (100%). The values are provided as mean ± SD from 2–3 independent experiments. For each treatment regimen, a nonlinear fit analysis of the resulting values was performed. (f) Glycosylation analysis of cellular β-Glu. Cell lysates of ABX, IFG or control-treated fibroblasts were subjected to Endo-H digestion and Western blot. Qualitative band analysis was repeated in two independent experiments: Endo-Hr, Endoglycosidase H-resistant; Endo-Hs, Endoglycosidase H-sensitive.