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Blood, Vol. 109, Issue 9, 4028-4037, May 1, 2007
Tumor suppressor PTEN is a physiologic suppressor of chemoattractant-mediated neutrophil functions
Blood Subramanian et al.
109: 4028
Supplemental materials for: Subramanian et al, Vol 109, Issue 9, 4028-4037
Files in this Data Supplement:
- Figure S1. The expression of PTEN protein is completely abolished in neutrophils isolated from PTENloxP/loxP;Cre+/− mice (JPG, 38.4 KB)
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(A) Mouse bone marrow was isolated from the femur and the tibia of 10-week-old mice. Bone marrow neutrophils are separated by centrifugation over a 3-layer Percoll gradient (78%/69%/52%). About 7 to 11 million cells per mouse were routinely obtained, and greater than 85% of them were morphologically mature neutrophils. Protein extracts were resolved on SDS-PAGE. PTEN protein was detected with a specific anti-PTEN antibody (Cell Signaling, Beverly, MA). (B) Relative amounts of PTEN protein in panel A were quantified using NIH ImageJ software (Bethesda, MD).
- Figure S2. Blood parameters for PTEN−/− and WT mice (JPG, 87.3 KB)
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Mouse blood samples (∼200 µL) were taken from the eye and analyzed. (A) Scatter plot of neutrophil, lymphocyte, monocyte, eosinophil, and basophil numbers per microliter of blood from 6 to 8 different PTEN−/− or WT mice. (B) Blood parameters represented as mean ± SD from 6 to 8 mice.
- Figure S3. F-actin fluorescence histograms (JPG, 64.3 KB)
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FACS histograms of wild-type (left) and PTEN−/− neutrophils (right) (0.5 × 106 cells) that were stimulated with 1 µM fMLP for 0, 1, 3, 5 minutes, fixed, and stained using 0.13 µg/mL fluorescein phalloidin. Geometric mean fluorescence (G-mean) for each histogram has also been indicated.
- Figure S4. Chemotaxis parameters for PTEN−/− and WT neutrophils (JPG, 96.1 KB)
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(A) Assume a cell migrating from point A to point C in response to a point source of chemoattractant. If n is number of frames captured, t is the time elapsed between each frame, and d is the distance between initial and final positions of the cell, the chemotaxis parameters can be calculated as follows. Average cell speed: (x1/t + x2/t +…)/n; average cell speed in the direction of the pipette tip is a measure of overall chemotaxis: (y1 − y2)/t + (y2 − y3)/t….)/n; average directionality toward pipette tip describes how straight the cell is moving toward the pipette: (y1 − y2)/x1 + (y2 − y3)/x2 ...)/n; chemotactic error is the average angular deviation from the direction of the pipette tip: ( ı + 2 + ...)/n, where 1 = Cos−1 (y1 − y2)/x1); cumulative cell speed: (x1 + x2 + …)/(n × t); cumulative cell speed in the direction of pipette tip: (y1 − y2) + (y2 − y3) +…. )/(n × t); directionality to pipette tip: (y1 − y2) + (y2 − y3) + .../(x1 + x2 + …); and cumulative directionality: d/(x1 + x2 + …). Other parameters such as acceleration, direction change, and persistence have been described elsewhere.1 (B) Chemotaxis parameters for PTEN−/− and WT neutrophils migrating toward a pipette source. Results are mean ± SE of 10 to 13 cells from 3 different movie sequences.
- Video 1. Bone marrow–derived neutrophils from PTEN−/− mice were plated on coverslips and uniformly stimulated with 50 nM fMLP (MOV, 1.37 MB)
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Image sequences were captured from multiple fields every 10 seconds. fMLP was added after the third frame was captured.
- Video 2. Bone marrow–derived neutrophils from WT mice were plated on coverslips and uniformly stimulated with 50 nM fMLP (MOV, 1 MB)
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Image sequences were captured from multiple fields every 10 seconds. fMLP was added after the fifth frame was captured.
- Video 3. PTEN−/− neutrophils displaying multiple pseudopodia as they migrate randomly in response to uniform fMLP (100 nM) (MOV, 168 KB)
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Images were captured every 10 seconds.
- Video 4. PTEN−/− neutrophils were plated on coverslips and exposed to a micropipette filled with 10 µM fMLP (MOV, 1.72 MB)
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Images were captured every 20 seconds.
- Video 5. WT neutrophils were plated on coverslips and exposed to a micropipette filled with 10 µM fMLP (MOV, 1.92 MB)
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Images were captured every 20 seconds.
REFERENCE
1. Soll D, Wessels D. Motion analysis of living cells. New York; Chichester England: Wiley-Liss; 1998.
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