Migration, actin polymerization, and CXCR4 expression of Ba/F3 cells expressing wild-type or ITD-Flt3 mutations (W51, W73, and W78). (A) Ba/F3 cells expressing wild-type Flt3 (▪) or ITD-Flt3 (W51, □; W73, ▨; and W78, ▦) were subjected to transwell migration in the presence or absence of a negative gradient of 100 ng/mL CXCL12 with no cytokine in the lower chamber. These mutations in the juxtamembrane domain of Flt3 cloned from the patients with AML induce IL-3–independent growth of Ba/F3 cells and the W51 and W78 ITD-Flt3 induce lethal myeloproliferative disease in a murine bone marrow transplantation assay.³²  Data represent mean ± SEM percentage migration for 3 experiments. *P < .001 compared with wild-type Flt3, and ‡P < .001 compared with 0/0 gradient. (B) Baseline actin polymerization of Ba/F3 cells expressing wild-type or ITD-Flt3 expressed as mean fluorescence intensity of phalloidin-FITC. Symbols indicate same information as in panel A. Data are the mean ± SEM of triplicate measurements from 1 of 2 experiments with similar results. *P < .001 compared with wild-type Flt3. (C) Migration of Ba/F3-Flt3 (wild type or ITD-Flt3) to 100 ng/mL rhCXCL12 or 10 ng/mL FL plus 100 ng/mL CXCL12. Symbols indicate same information as in panel A. Data are expressed as mean ± SEM percentage migration for 3 experiments. *P < .0001 compared with wild-type Flt3. (D, left) Expression of cell surface CXCR4 on Ba/F3 cells expressing ITD-Flt3 compared with cells expressing wild-type Flt3 analyzed by FACS. Histogram represents 1 of the 3 experiments with similar results. The open histogram with bold line shows isotype staining of cells expressing wild-type Flt3. The isotype for other cells was equivalent to cells expressing wild-type Flt3 (not shown). The right panel represents mean ± SEM percentage reduction of CXCR4 in cells expressing ITD-Flt3 compared with cells expressing wild-type Flt3 from 3 experiments. Symbols indicate same information as in panel A. *P < .001 compared with wild-type Flt3.