Physical association between FLT-1 receptor, hERG1, and β₁ integrin subunit in primary AML blasts: effect on cell migration. (A) Co-immunoprecipitation of FLT-1 and hERG1 in primary AML blasts. Cell lysates from 5 AML cases of different FAB (respectively, M4, M2, M6, M5, and M2; 3 of the cases correspond to samples reported in Table 1: 1 is A45, 3 is A64, and 5 is A53), cultured in the presence of serum were immunoprecipitated with anti FLT-1 antibody; blots were probed with anti pan hERG1 antibody (top), anti-β1 antibody (middle), and anti FLT-1 antibody (bottom). (B) Co-immunoprecipitation of FLT-1 and hERG1 in primary peripheral CD34⁺ cells. Cell lysates were obtained from CD34⁺ (lane 1) and CD34⁺ treated for 12 hours in vitro with IL-3, granulocyte colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor³¹  (lane 2); blot was probed with anti-pan hERG1 antibody (top) and anti-FLT-1 antibody (bottom). (C) Effect of hERG1 expression on primary AML blast migration. The migration assay was performed as reported in the legend to Figure 4 on leukemic blasts isolated from PB of 6 AML samples. Cells were treated as reported in the legend to Figure 4B. Left, hERG1⁺ samples (A34, A42, A7). Right, hERG1⁻ samples (A54, A10, A13). Values are reported as number of migrated cells/mL. The average number of migrated cells of hERG1⁺ samples (570 567 ± 83 749) was significantly higher (Student t test, P = .033*) than in hERG1⁻ samples (217 000 ± 72 266). The average of migrated cells of flt1-negative cells was: hERG1⁺ samples (182 500 ± 11 000), hERG1⁻ samples (120 000 ± 7400). (D) Effect of Way (1 μM) on migration of primary leukemia cells expressing or not expressing hERG1 channels. The migration assay was performed as described in the legend to Figure 4 on 3 hERG1⁺ leukemia samples (2 of which correspond to samples in panel A, on the left) and on one hERG1⁻ sample (A60; on the right). Cells were treated (▒) or not (□) with Way 123 398 (1 μM). Values are reported as percentage of the control and each determination represents the average of 3 individual chambers (bars ± SEM). *Statistically significant differences between samples indicated by the horizontal bars. * = P < .05, Student t test. Error bars represent standard deviation.