Figure 3
Figure 3. AMD3100 inhibits migration and transendothelial migration under static and shear flow conditions in BCWM.1. (A) Transwell migration assay was performed using no SDF-1 (▤ for control) or with the presence of SDF-1 30 nM (). BCWM.1 pretreated with AMD3100 20 μM for 2 hours inhibited migration by 50% compared with control in the presence of SDF-1 30 nM. Similarly, pretreatment of BCWM.1 with PTX 200 ng/mL for 2 hours inhibited migration in response to SDF-1 similar to AMD3100. Data show an average of 3 independent experiments (*P < .05, **P < .05). (B) Migration of CD19+ primary WM cells from 3 patients demonstrated significant migration in response to SDF-1 30 nM compared with control (no SDF-1). Pretreatment of the cells with AMD3100 20 μM for 2 hours showed significant reduction of migration compared with untreated cells (C) Transendothelial migration of BCWM.1. HUVECs were grown on the inner wells of the Boyden chamber migration wells until confluent. BCWM.1 untreated or pretreated with AMD3100 20 μM for 2 hours or CXCR4 knockdown BCWM.1 cell line were placed in the upper chambers with or without SDF-1 30 nM in the lower chambers. The number of cells that migrated to the lower chambers was counted after 4 hours. As shown, AMD3100 and CXCR4Kd showed decreased migration compared with control. The presence of SDF-1 did not induce significant increase in migration in the presence of endothelial cells (that secrete SDF-1), indicating that endothelial cells induce transmigration through the SDF-1 axis (*P = .04). (D) Shear flow chamber assay for rolling, firm adhesion to endothelial cells, and locomotion. Pretreatment of BCWM.1 cells with AMD3100 (20 μM for 2 hours) had no effect on rolling and firm adhesion (which are regulated by selectins) but had significant inhibition on the number of locomoting cells and the percentage of locomotion of adherent cells. Bars represent percent of untreated controls. Data show an average of 3 independent experiments (*P = .001, **P = .001). (E) Shear flow chamber assay for transendothelial migration showing the number of cells transmigrating as well as the percentage of cells transmigrating from the adherent and locomoting cell populations. Pretreatment of BCWM.1 cells with AMD3100 (20 μM for 2 hours) induced significant inhibition on transendothelial migration, specifically on the adherent cell population. Data show an average of 3 independent experiments (*P = .01, **P = .004).

AMD3100 inhibits migration and transendothelial migration under static and shear flow conditions in BCWM.1. (A) Transwell migration assay was performed using no SDF-1 (▤ for control) or with the presence of SDF-1 30 nM (). BCWM.1 pretreated with AMD3100 20 μM for 2 hours inhibited migration by 50% compared with control in the presence of SDF-1 30 nM. Similarly, pretreatment of BCWM.1 with PTX 200 ng/mL for 2 hours inhibited migration in response to SDF-1 similar to AMD3100. Data show an average of 3 independent experiments (*P < .05, **P < .05). (B) Migration of CD19+ primary WM cells from 3 patients demonstrated significant migration in response to SDF-1 30 nM compared with control (no SDF-1). Pretreatment of the cells with AMD3100 20 μM for 2 hours showed significant reduction of migration compared with untreated cells (C) Transendothelial migration of BCWM.1. HUVECs were grown on the inner wells of the Boyden chamber migration wells until confluent. BCWM.1 untreated or pretreated with AMD3100 20 μM for 2 hours or CXCR4 knockdown BCWM.1 cell line were placed in the upper chambers with or without SDF-1 30 nM in the lower chambers. The number of cells that migrated to the lower chambers was counted after 4 hours. As shown, AMD3100 and CXCR4Kd showed decreased migration compared with control. The presence of SDF-1 did not induce significant increase in migration in the presence of endothelial cells (that secrete SDF-1), indicating that endothelial cells induce transmigration through the SDF-1 axis (*P = .04). (D) Shear flow chamber assay for rolling, firm adhesion to endothelial cells, and locomotion. Pretreatment of BCWM.1 cells with AMD3100 (20 μM for 2 hours) had no effect on rolling and firm adhesion (which are regulated by selectins) but had significant inhibition on the number of locomoting cells and the percentage of locomotion of adherent cells. Bars represent percent of untreated controls. Data show an average of 3 independent experiments (*P = .001, **P = .001). (E) Shear flow chamber assay for transendothelial migration showing the number of cells transmigrating as well as the percentage of cells transmigrating from the adherent and locomoting cell populations. Pretreatment of BCWM.1 cells with AMD3100 (20 μM for 2 hours) induced significant inhibition on transendothelial migration, specifically on the adherent cell population. Data show an average of 3 independent experiments (*P = .01, **P = .004).

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