Figure 1. IFG increases N370S and L444P GCase activity in Gaucher patient-derived cells.
Panel A. N370S fibroblasts (DMN89.45) were incubated with the indicated concentrations of IFG tartrate for five days and GCase activity was directly measured in lysed cells as described in ‘Materials and Methods’. In the experiment shown, a concentration-dependent increase of approximately 2.5-fold was seen in GCase activity. The increase in GCase activity was found significant for a linear trend (one-way ANOVA), indicating a concentration-dependent effect. Panel B. L444P fibroblasts (GM07968) and LCLs (GS0501) were incubated with the indicated concentrations of IFG tartrate for five days and GCase activity was directly measured in lysed cells. In the experiments shown, a small but reproducible 1.3-fold increase in GCase activity was seen in fibroblast lysates (left panel), and a 3.5-fold increase was seen in LCL lysates (right panel). The increase in GCase activity measured in LCLs was found significant for a linear trend (one-way ANOVA). Summary data from the fibroblast and LCL cell lines shown here, as well as others, are presented in Table 1. Insets, GCase protein levels were increased in Gaucher fibroblasts and LCLs after five-day incubation with IFG, as directly measured by Western blotting (50 μg total protein per lane). Blots were probed with an anti-human GCase antibody and a β-actin antibody (loading control). The data shown are representative of three independent experiments. Panel C. Gaucher fibroblasts homozygous for L444P GCase (GM07968) were incubated for five days with the indicated concentrations of IFG tartrate. Cell lysates were then subjected to either glycoprotein- or GCase-enrichment using Con A- and immunocapture, respectively, as described in ‘Materials and Methods’. GCase activity was measured on the precipitated beads. In the experiments shown, concentration-dependent increases (approximately 2-fold) were seen in GCase activity. The increases were found significant for a linear trend (one-way ANOVA). Panel D. Gaucher fibroblasts homozygous for L444P GCase (GM07968) were incubated for five days with the indicated concentrations of IFG tartrate, followed by 24-hour washout (media only). GCase activity was measured directly in lysed cells. In the experiments shown, an approximately 1.7-fold increase was seen in L444P GCase activity after 24-hour washout. This increase was found significant for a linear trend (one-way ANOVA). In all panels, the data have been normalized to baseline (untreated) values and are representative of three or six independent experiments as indicated in Tables 1 and 2, with each point the mean±SEM of triplicate determinations. Statistically significant differences from untreated were determined using a two-tailed, unpaired student’s t-test with *p<0.05, **p<0.01, and ***p<0.001.