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Blood, Vol. 109, Issue 10, 4237-4244, May 15, 2007
A novel role for PECAM-1 in megakaryocytokinesis and recovery of platelet counts in thrombocytopenic mice
Blood Dhanjal et al.
109: 4237
Supplemental materials for: Dhanjal et al, Vol 109, Issue 10, 4237-4245
Files in this Data Supplement:
- Table S1. Circulating blood cell numbers and composition before and after platelet depletion in PECAM-1–/– mice (PDF, 19.1 KB) -
Total white cell count (WCC) numbers and differential leukocyte counts (×103/mm3) before (predepletion, n=16) and after (postdepletion, n=8) intraperitoneal injection with 2 µg/g anti-GP1b antibody. Cell numbers were determined by direct counting using a hemocytometer. *P<.05 for PECAM-1+/+ versus PECAM-1–/– (Student t test). All other comparisons are not statistically significant.
- Figure S1. Three-dimensional views of CXCR4 membrane localization in PECAM-1+/+ and PECAM-1–/– MKs (PDF, 310 KB) -
Stacked confocal fluorescence microscopic analyses using a specific antibody demonstrates marked polarization of CXCR4 in the representative PECAM-1+/+ MKs. Confocal fluorescence images were obtained using a Leica DMIRE 2 inverted microscope with a 40× objective. In the PECAM-1–/– MKs the receptor CXCR4 is distributed through all of the MK membrane.
- Figure S2. PECAM-1+/+ and PECAM-1–/– bone marrow cellular structure in high-resolution DIC and fluorescent images (PDF, 175 KB) -
Cryofrozen bone marrow longitudinal sections were prepared as described in the immunohistochemistry section of the Materials and methods. MKs were identified by FITC-CD41 and marrow sinusoidal vessels were identified by CD105/biotin/PE-streptavidin. The highlighted MKs (white arrows) are clearly visible in the high-resolution DIC images that correlate with the fluorescent images. A region of compact bone is visible in both PECAM-1+/+ and PECAM-1–/– sections in the top right corner of the fields.
- Video S1. Real-time chemotaxis of a PECAM-1+/+ MK exposed to an SDF1α gradient (MOV, 6.68 MB)
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Cell motility was assessed using the Dunn chamber on fibronectin-coated (10 µg/mL) coverslips. The Dunn chamber was placed in a humidified chamber on a heated stage at 37°C within an inverted microscope setup and time-lapse images were digitally captured every 60 seconds for 4 hours. The direction of the SDF1 chemokine gradient in this movie is from left to right of the screen and this representative MK clearly migrates in this direction with the formation of a clear pseudopod and polarization with a leading lamellipodial edge.
- Video S2. Real-time chemokinesis of a PECAM-1–/– MK exposed to an SDF1α gradient (MOV, 2.46 MB)
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Cell motility was assessed using the Dunn chamber on fibronectin-coated (10 µg/mL) coverslips. The Dunn chamber was placed in a humidified chamber on a heated stage at 37°C within an inverted microscope setup and time-lapse images were digitally captured every 60 seconds for 4 hours. The direction of the SDF1 chemokine gradient in this movie is from top left to bottom right of the screen. The representative PECAM-1–/– MK does not polarize and indeed retracts and extends in a number of random directions. It does not generate a clear pseudopod and as it moves in the various directions it generates a large lamellipodia, and ultimately there is a reduction in the overall displacement compared with the wild-type MK.
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