Overexpression of CD123 downregulates CXCR4 gene and protein expression in response to IL-3 and reduces chemotactic responses to SDF-1. Cells were cultured ±10 ng/mL mIL-3 for 48 to 72 hours posttransduction. (A) Gene expression of a panel of adhesion molecules and chemotactic receptors using RT-PCR. Expression is presented as fold change in response to IL-3 ± SEM, n = 3. (B) Flow cytometry analysis of surface CXCR4 expression reveals specific downregulation in cells overexpressing CD123 in response to IL-3 (black, negative; blue, CD123-high-WT no IL-3; red, +CD123-high-WT + IL-3). (C) CXCR4 gene expression changes in response to IL-3 were analyzed in FL containing CD123-low (EV), CD123-high-WT, or CD123-high-APIA. Data were normalized to β-actin and represented as foldchange in response to IL-3 stimulation ± SEM, n = 3. (D) TF-1 cells were cultured in IL-3 or GM-CSF and expression of CXCR4 measured by flow cytometry (black, negative; blue, no IL-3; red, +IL-3). (E) SDF-1–induced migration demonstrates reduced chemotactic response with CD123 overexpression, which can be rescued by addition of muCXCR4. Migration was analyzed using a transwell migration assay and migrated cells were counted using flow cytometry and fluorospheres. Data are normalized to a blank sample and are presented as mean migration relative to 10% FBS ± SEM, n = 3. Statistical analysis: #nonsignificant difference, *P < .05, **P < .01, ***P < .001.