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Blood, Vol. 107, Issue 3, 870-879, February 1, 2006
cAMP-induced PKC activation increases functional CXCR4 expression on human CD34+ hematopoietic progenitors
Blood Goichberg et al.
107: 870
Supplemental materials for: Goichberg et al, Vol 107, No 3, 870-879
Files in this Data Supplement:
- Supplemental Methods (PDF, 14.2 KB)
- Figure S1. Effect of signal transduction inhibitors on cAMP-induced membranal CXCR4 expression in CD34+ cells (JPG, 36.6 KB)
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CD34+ cells were incubated for 18 hours, either untreated (CTRL) or treated with 500 µM dbcAMP (cAMP), without (—) or in the presence of the following agents: PSζ (10 µM), H-89 (10 and 30 µM), KT5720 (0.5 and 1 µM), NF-κBSN50 (3, 12, and 18 µM), wortmannin (50 µM), LY294002 (10 µM), and Y27632 (20 µM). Cells were then analyzed by flow cytometry. Results shown are representative of at least 2 independent experiments.
- Figure S2. Transient effect of cAMP treatment on the BM homing potential and CXCR4 expression (JPG, 42.6 KB)
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(A) MPBL and CB CD34+ cells were treated with 500 µM dbcAMP (cAMP) for the time periods indicated in parentheses or were left untreated (CTRL). Numbers of CD45+ human cells in the BM of recipient mice were assayed by flow cytometry 24 hours after transplantation. Data represent mean ± SD values for each experiment, with 2 mice per treatment in each of the experiments (exp). (B) Reversal of the increase in CXCR4 membranal expression following cAMP removal from the growth medium. G2 cells were incubated for 24 hours, either untreated (CTRL) or treated with 500 µM cAMP, then either washed in excess growth medium (wash) or left untreated. They were then further cultured for the specified time periods. CXCR4 expression was determined by flow cytometry. Results are mean ± SD of 3 independent experiments. P values indicate statistically significant differences between cAMP-treated washed and unwashed cells.
- Figure S3. Priming with the cAMP-elevating agents antagonizes SDF-1—induced responses in CD34+ human hematopoietic progenitors (JPG, 50.2 KB)
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CB CD34+ cells were incubated for 30 minutes either untreated or treated with EtOH vehicle (CTRL), 10+nM PGE2, or 500 µM dbcAMP (cAMP). (A) Immunocytochemical analysis of cells treated as described above, adhered for 30 minutes to hyaluronic acid-coated glass coverslips in the absence (—), or presence (+) of 25 nM SDF-1, and stained with phalloidin for polymerized actin. Bar represents 50 µm. A typical cell for each treatment is shown in the insert (original magnification ×100). (B) Representative flow cytometry analysis of changes in the level of filamentous actin in cells treated with 37 nM SDF-1 for the indicated time periods and fixed and stained as described above. Results are shown as percent change relative to the mean intensity of CTRL cells (100%). (C) PKCζ phosphorylation status in G2 cells preincubated for 30 minutes without (CTRL) or with 500 µM cAMP and stimulated for additional 10 minutes with 25 nM SDF-1 (+), or left untreated (—). Data are expressed as mean ± SD of 2 independent experiments. (D) Representative flow cytometry analysis of membranal CXCR4 expression in CB CD34+ cells incubated for 30 minutes without (CTRL) or with 500 µM cAMP. IgG indicates secondary IgG only-labeled cells.
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