Supplementary Materials

Materials and Methods:

Proliferation assays:

To measure responsiveness to angiogenic growth factors, cells were seeded at a density of 104 cells/cm2. The following day cells were starved with 1% FBS in DMEM overnight. Cultures were then treated with various concentrations of bFGF or VEGF (Invitrogen) in the same medium for 2 days. At the end of treatment, cells were trypsinized and cell numbers were counted.

Tubulogenesis assay:

Matrigel matrix (BD Biosciences) was added to 96-well plates and was allowed to polymerize at 37°C for 30 min. Cells suspended in EGM-2MV medium were seeded onto the matrix at a density of 2x104 cells/well. Tubulogenesis was evaluated on the following day. IMFE1 cells (Shen et al 2007) were used as positive control.

Immunofluorescence staining:

Immunostaining was performed as described (Shen et al 2007) with modifications. After fixation, the cells were permeabilized with 0.1% saponin in 10% normal goat serum for 1 h at room temperature. Saponin (0.05%) was included in dilution buffer (5% normal goat serum in PBS) for primary and secondary antibodies. Primary antibodies used were mouse monoclonal to SV40 T antigen (Pab108, Santa Cruz) and Gb3 (Seikagaku, Tokyo, Japan), and rabbit polyclonal to vWF (DAKO) and sortilin (abcam).

Western blot:

Western blot was performed as described (Shen et al 2007). NuPAGE Bis-Tris 4-12% or Tris-Acetate 3-8% gels (Invitrogen) were used for protein separation. Primary antibodies used were rabbit polyclonal to VCAM-1 (Santa Cruz Biotech) and sortilin (abcam), rabbit monoclonal to M6PR (Cell Signaling), and rat monoclonal to MR (Santa Cruz Biotech). GAPDH (Santa Cruz Biotech) was used as loading control.

Reference:

Shen JS, Meng XL, Schiffmann R, Brady RO, Kaneski CR (2007) Establishment and characterization of Fabry disease endothelial cells with an extended lifespan. Mol Genet Metab 92: 137-144.

Supplementary Figure 1. β-glucocerebrosidase activities in CBE-treated FMEC2

FMEC2 were treated with 400 μM CBE for 0, 1, 3, 24 and 48 h, and β-glucocerebrosidase activities were measured using the fluorimetric method (n=4).

Supplementary Figure 2. Uptake of α-gal A preparations in FMEC2

(a) Uptake of agalsidase alfa and moss-aGal in FMEC2 at various concentrations. Cells were incubated for 4 h. Data are means of duplicates. (b) Uptake rates of agalsidase alfa and moss-aGal (10 μg/ml) in FEMC2 (n=3).