CD44s directly interacts with CXCR4 upon CXCL12 stimulus. (A) OCI-AML3 cells were treated with CXCL12 and increasing concentrations of HA. (B) OCI-AML3 cells transiently transfected with CD44 siRNA or control siRNA were treated with CXCL12 (200 ng/mL) and/or HA (200 μg/mL). ERK phosphorylation was detected using a phospho-ERK-specific antibody by western blot. A representative picture is shown. Data from 3 independent experiments were quantified. Three independent experiments were quantified (Kruskal-Wallis test; *P < .05; **P < .01). (C) Upper rows: HEK293T cells were transfected with a plasmid encoding CXCR4 fused with the N-terminal fragment of the Venus fluorescent protein (CXCR4-VN) and with a plasmid encoding CD44 standard fused with the C-terminal domain of Venus (CD44s-VC). The transfected cells were either left uninduced or were induced with CXCL12, HA, or a combination of both as indicated. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). The bimolecular fluorescence complementation assay signal (yellow) was visualized by confocal microscopy (LSM800 Zeiss microscope 63× objective; scale bar, 20 μm). Mean corrected total cell fluorescence (CTCF) of each cell was analyzed from 4 independent experiments (n = 200 cells). Lower rows: HEK293T cells transfected as indicated above were treated with AMD3100 (5 µM for 10 minutes) or left untreated and subsequently induced with CXCL12 (200 ng/mL for 10 minutes) or left uninduced. Mean CTCF of approximately 450 cells of each condition from 2 independent experiments were analyzed by 1-way ANOVA. *P < .05; **P < .005; ***P < .0005; ****P < .0001.