Figure 2
Figure 2. CXCR4 expression on the surface of pML cells and SDF-1α–induced CXCR4 translocation. (A) Flow cytometric analysis of cell-surface expression of CXCR4 in pML cells. CXCR4 was detected by incubating cells with PE-conjugated rat anti-CXCR4 antibody. Red line represents CXCR4 expression, and gray area represents the result of staining with isotype-matched control antibody. (B) CXCR4 expression on pML cell surface. Cells were treated in the presence (blue dot line) or absence (red line) of 100 ng/mL SDF-1α. After a brief wash, cells were incubated with the same antibody as used in Figure 2A. The gray area represents staining with isotype-matched control antibody. (C) Total cellular protein level of CXCR4 in the presence (+) or absence (−) of SDF-1α (100 ng/mL) for 5 minutes. (D) SDF-1α–induced phosphorylation of ERK1/2 in pML cells. pML cells were treated before lysis with 100 ng/mL SDF-1α for the indicated times. Cell lysates were analyzed by immunoblotting analysis with anti–phospho-ERK, –total ERK, –phospho-AKT, –total AKT, –phospho-PI3K, –phospho-P38, –phospho-IκBα, –total IκBα, or –β-actin antibodies. (E) Expression levels of cellular surface CXCR4 in pML cells after treatment with AMD3100. Cells were pretreated with 25 μg/mL AMD3100 for 1 hour, and then stimulated with SDF-1α for 5 minutes (black dot line). Cells were also stimulated exclusively with SDF-1α (blue dot line) or were untreated (red line). The gray area represents staining with isotype-matched control antibody. (F) Expression of total CXCR4 protein with (+) or without (−) AMD3100 treatment. The lysates from treated cells were analyzed by immunoblotting with anti–mouse/human CXCR4 polyclonal antibody. (G) Phosphorylation of ERK1/2 in pML cells with or without AMD3100 treatment. After pretreatment with AMD3100 (25 μg/mL) for 1 hour, cells were stimulated with SDF-1α for the indicated times (0, 5, 30, 60, and 120 minutes). The cellular lysates were analyzed by immunoblotting. In the lane at the right, the cells received no AMD3100 treatment but were stimulated with SDF-1α. (H) Migration assay of pML cells using AMD3100 at various concentrations (0, 0.25, 0.5, and 1.25 μg/mL). pML cells were preincubated for 60 minutes with AMD3100 at the indicated concentrations, and then subjected to the migration assay in the presence of 100 ng/mL SDF-1α. The data were presented as a relative ratio of migrating cells: the number of migrated cells in the presence of AMD3100/the number of nontreated cells. These results were confirmed by 3 independent experiments. The data are presented as mean values ± SD. *P < .05. **P < .01.

CXCR4 expression on the surface of pML cells and SDF-1α–induced CXCR4 translocation. (A) Flow cytometric analysis of cell-surface expression of CXCR4 in pML cells. CXCR4 was detected by incubating cells with PE-conjugated rat anti-CXCR4 antibody. Red line represents CXCR4 expression, and gray area represents the result of staining with isotype-matched control antibody. (B) CXCR4 expression on pML cell surface. Cells were treated in the presence (blue dot line) or absence (red line) of 100 ng/mL SDF-1α. After a brief wash, cells were incubated with the same antibody as used in Figure 2A. The gray area represents staining with isotype-matched control antibody. (C) Total cellular protein level of CXCR4 in the presence (+) or absence (−) of SDF-1α (100 ng/mL) for 5 minutes. (D) SDF-1α–induced phosphorylation of ERK1/2 in pML cells. pML cells were treated before lysis with 100 ng/mL SDF-1α for the indicated times. Cell lysates were analyzed by immunoblotting analysis with anti–phospho-ERK, –total ERK, –phospho-AKT, –total AKT, –phospho-PI3K, –phospho-P38, –phospho-IκBα, –total IκBα, or –β-actin antibodies. (E) Expression levels of cellular surface CXCR4 in pML cells after treatment with AMD3100. Cells were pretreated with 25 μg/mL AMD3100 for 1 hour, and then stimulated with SDF-1α for 5 minutes (black dot line). Cells were also stimulated exclusively with SDF-1α (blue dot line) or were untreated (red line). The gray area represents staining with isotype-matched control antibody. (F) Expression of total CXCR4 protein with (+) or without (−) AMD3100 treatment. The lysates from treated cells were analyzed by immunoblotting with anti–mouse/human CXCR4 polyclonal antibody. (G) Phosphorylation of ERK1/2 in pML cells with or without AMD3100 treatment. After pretreatment with AMD3100 (25 μg/mL) for 1 hour, cells were stimulated with SDF-1α for the indicated times (0, 5, 30, 60, and 120 minutes). The cellular lysates were analyzed by immunoblotting. In the lane at the right, the cells received no AMD3100 treatment but were stimulated with SDF-1α. (H) Migration assay of pML cells using AMD3100 at various concentrations (0, 0.25, 0.5, and 1.25 μg/mL). pML cells were preincubated for 60 minutes with AMD3100 at the indicated concentrations, and then subjected to the migration assay in the presence of 100 ng/mL SDF-1α. The data were presented as a relative ratio of migrating cells: the number of migrated cells in the presence of AMD3100/the number of nontreated cells. These results were confirmed by 3 independent experiments. The data are presented as mean values ± SD. *P < .05. **P < .01.

Close Modal
This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal