Cell migration in response to FLT-1and integrin stimulation of human AML cell lines: role of hERG1 channels. (A) Analysis of cell migration in FLG 29.1 cells. Cells were allowed to migrate through BSA-coated filters (□) and on FN-coated filters in the presence of VEGF₁₆₅ (100 ng/mL), or PlGF (50 ng/mL). Cell migration was carried out at 37°C and 5% CO₂ for 18 hours as detailed in “Patients, materials, and methods; Migration assay.” Values are reported as number of migrated cells/mL and represent means of 4 experiments, each performed in triplicate. Results shown are means (± SEM). The following inhibitors were used, at the final concentrations reported in parentheses: the hERG1 specific blocker Way (1 and 40 μM); the specific hERG1 blocker E4031 (1μM); tetra-ethyl-ammonium (used at 5 mM, a concentration known to block voltage-dependent K⁺ channels other than hERG1); C. toxin, a blocker of Ca²⁺-dependent K⁺ channels (1 μM); the anti FN-R antibody, known to block all the β₁ containing integrins²⁰  (1:50); the MAPK inhibitor LY (10 μM); the PI3K inhibitor PD (30 μM). When needed, cells were pretreated with ion channel inhibitors at 37°C for 15 minutes as reported in “Patients, materials, and methods; Treatment with channels inhibitors.” FLG 29.1 were also treated for 48 hours with Akt-siRNA and migration was assessed for a further 18 hours. Control scrambled siRNAs were used as reported in “Patients, materials and methods; Silencing of Akt by small interfering RNAs.” *Statistically significant differences between samples are indicated by the horizontal bars and are P < .05, Student t test. Inset, WB analysis of Akt expression levels in cells treated with Akt-siRNA or scrambled-siRNA. (B) Effects of various treatments on FLG 29.1 cell proliferation/survival. Cells, treated as in panel A, were incubated in 96-well cell culture plates for 18 hours. At the end of incubation, the WST reagent was added, and absorbance was measured. Data are reported as percentage of the control and represent mean (± SEM) of 3 different experiments, each performed in triplicate. (C) Effect of VEGF and integrin stimulation, as well as of hERG1 blockers on migration of various AML cell lines. Cell migration and treatments were performed as in panel A. Results shown are means (± SEM). *Statistically significant differences between samples are indicated by the horizontal bars and are P < .05, Student t test. The correlation between the amount of leukemia cells stimulated to migrate by VEGF plus FN (normalized on the amount of migrated cells in control conditions) and the amount of FLT-1/hERG1/β₁ complex in cells stimulated by VEGF plus FN (normalized on the amount of the complex in cells treated with BSA, taken from Figure 2C) was determined by regression analysis (P = .02).