Effect of the ROCK inhibitor fasudil on malignant and nonmalignant hematopoietic cells. (A) Proliferation kinetics of untreated cells from the indicated donors over 96 hours of suspension culture (fold expansion from baseline to the end of the suspension-culture period is given). To allow for the cross-donor comparisons (B), all results obtained after 96 hours of fasudil treatment (40 μM), were normalized to wells containing vehicle-treated cells of each respective donor. The index patient AML2 (black) is shown in comparison with 3 other AML donors (AML24, AML26, and AML29; gray) and 3 normal donors (N16, N9, and N10; white). Means and standard deviations of experiments done in triplicate are presented. (C-H) Results of experiments done with leukemic and nonmalignant hematopoietic progenitors in LTC-IC. Representative microscopic images before (day 0) and after 4 days of treatment with carrier (control) or active drug (fasudil) at 40 μM concentration are shown for AML2 (C), AML22 (E), and normal CD34⁺ HSPCs isolated from volunteer donors (G). Red lines highlight cobblestone areas. Note the disappearance of cobblestone cells in the fasudil-treated sample in the AML cells but not in normal HSPCs. All images were taken using the Celigo cytometer according to the manufacturer’s instructions; native live cell imaging. Quantification of the changes of cobblestone numbers after 4 days of treatment with fasudil or carrier (control) in AML2 (D), AML22 (F), and normal CD34⁺ HSPCs (H). Means and standard deviations of 4 to 8 replicates are shown. All cobblestones present within each well were counted.