Figure 5.
Figure 5. Effects of preculture of UCB CD34+ and Ba/F3-Flt3 cells with FL on migration to CXCL12, CXCR4 expression, and phosphorylation of intracellular signaling molecules. (A) Flow cytometric analysis of expression of cell surface CXCR4 on UCB CD34+ cells cultured with or without 100 ng/mL FL for 48 hours in 20% HI-FBS using antihuman CXCR4 antibody (clone 12G5). The filled and open histograms show isotype and CXCR4 staining, respectively. The top panel shows CXCR4 expression in cells incubated without FL, and the bottom panel shows cells pretreated with FL. The histogram is representative of 6 samples analyzed. (B) Percentage migration of UCB CD34+ cells cultured without (▪) or with 100 ng/mL FL (□) for 48 hours to CXCL12. After culture, cells were washed and subjected to migration assay with 100 ng/mL CXCL12. The panel represents 1 of the 3 experiments with similar results. *P < .001 compared with control cells without FL pretreatment. Data expressed are mean ± SEM. (C) Expression of cell surface CXCR4 on Ba/F3 cells or Ba/F3-Flt3 cells cultured with FL. Exponentially growing Ba/F3 cells or Ba/F3-Flt3 cells were cultured with (+) or without (–) 100 ng/mL rhFL in the presence of IL-3 for 24 hours. CXCR4 expression was analyzed by flow cytometry using antimouse CXCR4 antibody (clone 2B11). The filled and open histograms show isotype and CXCR4 staining, respectively. The data represent 1 of 3 experiments. (D) The graph shows mean ± SEM percent migration of Ba/F3-Flt3 cells to CXCL12 precultured without (▪) or with FL (□) for 24 hours from 3 experiments. *P < .001 compared with control cells without FL pretreatment. (E, top) Phosphorylation of MAPKp42/p44, Akt, CREB, and ATF-1 upon stimulation with 100 ng/mL CXCL12 in Ba/F3-Flt3 cells preincubated with (+) or without (–) FL. Following culture as described in panel C, cells were washed extensively and incubated with 100 ng/mL CXCL12 for up to 15 minutes. Protein phosphorylation relative to total protein was quantified by densitometry and shown in the bottom panel. ▵ indicates –FL; •, +FL.

Effects of preculture of UCB CD34+ and Ba/F3-Flt3 cells with FL on migration to CXCL12, CXCR4 expression, and phosphorylation of intracellular signaling molecules. (A) Flow cytometric analysis of expression of cell surface CXCR4 on UCB CD34+ cells cultured with or without 100 ng/mL FL for 48 hours in 20% HI-FBS using antihuman CXCR4 antibody (clone 12G5). The filled and open histograms show isotype and CXCR4 staining, respectively. The top panel shows CXCR4 expression in cells incubated without FL, and the bottom panel shows cells pretreated with FL. The histogram is representative of 6 samples analyzed. (B) Percentage migration of UCB CD34+ cells cultured without (▪) or with 100 ng/mL FL (□) for 48 hours to CXCL12. After culture, cells were washed and subjected to migration assay with 100 ng/mL CXCL12. The panel represents 1 of the 3 experiments with similar results. *P < .001 compared with control cells without FL pretreatment. Data expressed are mean ± SEM. (C) Expression of cell surface CXCR4 on Ba/F3 cells or Ba/F3-Flt3 cells cultured with FL. Exponentially growing Ba/F3 cells or Ba/F3-Flt3 cells were cultured with (+) or without (–) 100 ng/mL rhFL in the presence of IL-3 for 24 hours. CXCR4 expression was analyzed by flow cytometry using antimouse CXCR4 antibody (clone 2B11). The filled and open histograms show isotype and CXCR4 staining, respectively. The data represent 1 of 3 experiments. (D) The graph shows mean ± SEM percent migration of Ba/F3-Flt3 cells to CXCL12 precultured without (▪) or with FL (□) for 24 hours from 3 experiments. *P < .001 compared with control cells without FL pretreatment. (E, top) Phosphorylation of MAPKp42/p44, Akt, CREB, and ATF-1 upon stimulation with 100 ng/mL CXCL12 in Ba/F3-Flt3 cells preincubated with (+) or without (–) FL. Following culture as described in panel C, cells were washed extensively and incubated with 100 ng/mL CXCL12 for up to 15 minutes. Protein phosphorylation relative to total protein was quantified by densitometry and shown in the bottom panel. ▵ indicates –FL; •, +FL.

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