Fig. 4.

Plerixafor-induced proliferation does not require substantial CXCR4 surface expression but is associated with AKT activation. a CXCL12 and plerixafor bind to the CXCR4 receptor. TC-32 and CADO-ES1 cells were grown in standard conditions and treated with CXCL12 (100 ng/ml) and/or plerixafor (1 μM) for 12 h before flow cytometric analysis of CXCR4 signal with a 12G5-CXCR4 antibody. b Significant reduction in CXCR4 mRNA (left panel) and surface protein expression (middle and right panel). Cells were transfected with CXCR4-targeting siRNA (siCXCR4) or non-silencing control (siCtrl) and analyzed by real-time quantitative PCR and flow cytometry after 48 h. c Proliferative plerixafor effects are maintained despite significant reduction in CXCR4 surface expression. 48 h after siRNA transfection, cells from (b) were treated with plerixafor for another 72 h. Relative cell number was measured by WST-1 assay. All graphs of this figure represent means ± SD of three independent experiments. d AKT is activated in response to plerixafor in CXCR4-high and -low cell lines. Cells grown in standard conditions were treated with plerixafor (1 μM) for 12 h or remained untreated. Whole cell lysates were analyzed by Western blotting. Actin was loading control