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CCR1 drives dissemination of multiple myeloma plasma cells
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Figure 6. CCX9588 treatment prevents activation of CCR1 signaling in multiple myeloma plasma cells and their migration towards CCL3 in vitro. (A) Relative num- ber of OPM2-EV-1 cells was assessed after 72 hours of treatment with 10 nM to 1 mM CCX9588 (all containing 0.01% dimethyl sulfoxide [DMSO)], or 0.01% DMSO vehicle alone. (B) Viability of OPM2-EV-1 cells was assessed after 72 hours of treatment with 10 nM to 1 mM CCX9588 (all containing 0.01% DMSO), or 0.01% DMSO vehicle alone. (C) Relative number of RPMI-8226-luc cells was assessed after 72 hours of treatment with 10 nM-1 mM CCX9588 (all containing 0.01% DMSO), or 0.01% DMSO vehicle alone. (D) Viability of RPMI-8226-luc cells was assessed after 72 hours of treatment with 10 nM to 1 mM CCX9588, or 0.01% DMSO vehicle by WST-1 assay. (E) OPM2-EV-1 cells were treated with CCX9588 (10 nM to 1 μM) or media alone for 24 hours, and cells were stimulated with 100 ng/mL rhCCL3 for 5 minutes. Cells were lysed and western blotting was performed with antibodies against p-AKT, p-ERK1/2, total AKT and total ERK. Hsc70 was used as a loading control. A representative of three experiments is shown. (F) RPMI-8226-luc cells were treated with CCX9588 (10 nM to 1 μM) or media alone for 24 hours, and cells were stimulated with 100 ng/mL rhCCL3 for 5 minutes. Cells were lysed and western blotting was performed with antibodies against p-AKT, p-ERK1/2, total AKT and total ERK. Hsc70 was used as a loading control. A representative of two experiments is shown. (G) OPM2-EV-1 cells were treated with 1 mM CCX9588 or 0.01% DMSO vehicle control for 24 hours and migrated towards 100 ng/mL rhCCL3 or media alone. (H) RPMI-8226-luc cells were treated with 100 nM CCX9588 or 0.01% DMSO vehicle control for 24 hours and migrated towards 100 ng/mL rhCCL3 or media alone. Graphs depict mean ± standard error of the mean of three or more independ- ent experiments (A to D, G to H). **P<0.01, *P<0.05, one-way ANOVA with Tukey’s multiple comparisons test (C) two-way ANOVA with Sidak’s multiple comparisons test (G-H).
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