Figure 4
Figure 4. Regulation of migration by inhibitors of the CXCR4 signaling pathway. (A) Migration assay using the CXCR4 inhibitor AMD3100 (0-100 μM). AMD3100 (10 μM) induced 70% inhibition of migration compared with control (P = .03). All wells contained 20 nM SDF-1 in the lower chambers. (B) Migration assay using the anti-CXCR4 antibody MAB171. Serial concentrations of MAB171 (0-400 μg/mL) inhibited migration in a dose-dependent fashion. Anti–CXCR4 MAB171 (10 μM) inhibited migration to 53%, and anti-CXCR4 MAB171 (200 μM) inhibited migration to 35% compared with control (P = .007). IgG control antibody (400 μg/mL) was used in the control well. (C) Transwell migration assay of MM.1S mock and MM.1S infected with CXCR4 shRNA (CXCR4 knockdown cells) in the presence or absence of SDF-1 (30 nM). CXCR4 knockdown cells migrated only to 43% of control, similar to cells not exposed to SDF-1 (ie, 60% reduction in migration compared with control mock cells treated with SDF-1). Control was mock cells treated with SDF-1. (D) Transwell migration assay of MM.1S in the presence or absence of the anti-CXCR4 antibody MAB171 (200 μg/mL). SDF-1 (30 nM) was placed in the lower chamber in the control wells. Bone marrow supernatant from patients with MM (2 patients) was placed in the lower chambers of the other wells. The BM supernatant bar represents the mean percentage migration of MM.1S compared with control. MM.1S migrated in response to BM supernatant (75.6%) compared with control. MAB171 resulted in 36% inhibition of migration in the SDF-1 chambers and 52.5% inhibition of migration in the chambers with BM supernatant. (E) MTT growth inhibition assay using MM cell lines treated with serial concentrations of AMD3100. AMD3100 did not inhibit survival compared with control. (F) Migration assay in MM.1S treated with inhibitors of pathways downstream of CXCR4: PTX, LY294002, rapamycin, PD098059, combination LY 294002 and PD098059, and p38 MAPK inhibitor SB203580. SDF-1 (30 nM) was placed in the lower chambers. PTX (50 ng/mL) significantly inhibited migration to 30% compared with control in the presence of 30 nM SDF-1 (P = .004). The PI3K inhibitor LY294002 and the MEK inhibitor PD098059 inhibited migration by 57% and 58%, respectively. Rapamycin downstream of PI3K demonstrated results similar to those of LY294002. The combination of the PI3K and ERK/MAPK inhibitors was not additive (59%), indicating that both signal through the same pathway. The p38 MAPK inhibitor SB203580 (SB) did not inhibit migration in response to SDF-1.

Regulation of migration by inhibitors of the CXCR4 signaling pathway. (A) Migration assay using the CXCR4 inhibitor AMD3100 (0-100 μM). AMD3100 (10 μM) induced 70% inhibition of migration compared with control (P = .03). All wells contained 20 nM SDF-1 in the lower chambers. (B) Migration assay using the anti-CXCR4 antibody MAB171. Serial concentrations of MAB171 (0-400 μg/mL) inhibited migration in a dose-dependent fashion. Anti–CXCR4 MAB171 (10 μM) inhibited migration to 53%, and anti-CXCR4 MAB171 (200 μM) inhibited migration to 35% compared with control (P = .007). IgG control antibody (400 μg/mL) was used in the control well. (C) Transwell migration assay of MM.1S mock and MM.1S infected with CXCR4 shRNA (CXCR4 knockdown cells) in the presence or absence of SDF-1 (30 nM). CXCR4 knockdown cells migrated only to 43% of control, similar to cells not exposed to SDF-1 (ie, 60% reduction in migration compared with control mock cells treated with SDF-1). Control was mock cells treated with SDF-1. (D) Transwell migration assay of MM.1S in the presence or absence of the anti-CXCR4 antibody MAB171 (200 μg/mL). SDF-1 (30 nM) was placed in the lower chamber in the control wells. Bone marrow supernatant from patients with MM (2 patients) was placed in the lower chambers of the other wells. The BM supernatant bar represents the mean percentage migration of MM.1S compared with control. MM.1S migrated in response to BM supernatant (75.6%) compared with control. MAB171 resulted in 36% inhibition of migration in the SDF-1 chambers and 52.5% inhibition of migration in the chambers with BM supernatant. (E) MTT growth inhibition assay using MM cell lines treated with serial concentrations of AMD3100. AMD3100 did not inhibit survival compared with control. (F) Migration assay in MM.1S treated with inhibitors of pathways downstream of CXCR4: PTX, LY294002, rapamycin, PD098059, combination LY 294002 and PD098059, and p38 MAPK inhibitor SB203580. SDF-1 (30 nM) was placed in the lower chambers. PTX (50 ng/mL) significantly inhibited migration to 30% compared with control in the presence of 30 nM SDF-1 (P = .004). The PI3K inhibitor LY294002 and the MEK inhibitor PD098059 inhibited migration by 57% and 58%, respectively. Rapamycin downstream of PI3K demonstrated results similar to those of LY294002. The combination of the PI3K and ERK/MAPK inhibitors was not additive (59%), indicating that both signal through the same pathway. The p38 MAPK inhibitor SB203580 (SB) did not inhibit migration in response to SDF-1.

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