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Blood, Vol. 109, Issue 8, 3417-3423, April 15, 2007
A novel PAX5-ELN fusion protein identified in B-cell acute lymphoblastic leukemia acts as a dominant negative on wild-type PAX5
Blood Bousquet et al.
109: 3417
Supplemental materials for: Bousquet et al, Vol 109, Issue 8, 3417-3423
Supplemental materials and methods Samples. Immune phenotype had been previously studied, and the patients P1, C3, and C5 were classified respectively as pre-B3 (CD10+ Cµ+), pre-B3 and pre-B2 (CD10+ Cµ–), according to the European Group for the Immunological Characterization of Leukemias (EGIL) classification. Normal peripheral blood lymphocytes, isolated on density gradient, were used as control for quantitative membrane expression of CD19. Quantification of CD19. We used quantitative direct labeling flow cytometry with standard fluorescent beads (DAKO fluorospheres) on a FACSCALIBUR (Becton Dickinson) to compare CD19 expression on 3 patients with pre-B ALL (P1, C3, and C5). Fluorospheres. The DAKO fluorospheres are a set of 6 premixed beads, with 5 sets of beads corresponding, respectively, to a density of 1.5 × 103, 4.4 × 103, 14.5 × 103, 43.8 × 103, and 131.2 × 103 molecule equivalent fluorochrome (MEF R-phycoerythrin RPE), and 1 nonfluorescent set used as a blank control. FACS settings. Photomultiplier tube (PMT) voltage was determined to allow us to read the fluorescence of the beads in a logarithmic amplification over a 3.5- to 4-decade range, and then remained unchanged for the entire experiment. The standard regression line between MEF RPE and mean fluorescence intensity (MFI) was drawn using the value of MFI for each kind of bead. Immunofluorescence assay. Briefly, after thawing, cell viability was assessed. Each cell suspension contained more than 80% of viable cells (determined by trypan blue exclusion). Suspensions of 106 cells were then incubated 30 minutes with a mouse monoclonal anti-CD19 coupled with a phycoerythrin fluorochrome (Dako clone HD37) under saturating concentrations, washed once, and immediately analyzed, gating the lymphoblastic cells.
Files in this Data Supplement:
- Table S1. CD19 protein quantification (PDF, 9.36 KB) -
The levels of CD19 protein (expressed in MEF RPE) were not significantly different between the patient (P1) carrying the PAX5-ELN fusion gene and 2 PAX5-ELN–negative patients with B-ALL (C3 and C5).
- Figure S1. Luciferase activity control (PDF, 13.2 KB) -
The plasmids luc-CD19 (5 µg) and the reference pRL-CMV (0.4 µg; transfection efficiency control) were transfected together into HeLa cells with increasing amounts of the pcDNA3-PAX5. Luciferase activity of 3 independent transfection experiments was normalized to the measured Renilla activities and are shown as average values relative to the basal activity observed with pcDNA3 alone. The increasing amount of vector has no effect on the luciferase activity.
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