2D drug screening in AML-MSCs identifies lercanidipine. (A) Cell proliferation by ATP in 2D culture after treatment with 480 chemically active compounds used at 10 μM in AML-MSCs (n = 6). Compounds that reduced cell proliferation more than 50% were tested on h-MSCs (n = 2) and AML cell lines (HL-60 and ML-2). Cell proliferation ranged from 0% (red) to 100% (pink), relative to DMSO value. Treatments were performed in triplicate. (B) Cell proliferation by ATP in 2D culture of AML-MSCs (n = 6), h-MSCs (n = 3), AML cell lines (n = 2), primary AML cells (n = 6), and cord blood–derived CD34⁺ cells (n = 2) after 48 hours of lercanidipine treatment, relative to DMSO value. Treatments were performed in triplicate; dotted line represents 50% of cell proliferation. (C) Immunofluorescence staining of CaV1.2 (green) expression, F-actin (red), and DAPI nuclear counterstain (blue) in MSCs cells in 2D culture; ×40 magnification; scale bar, 10 µm, n = 6. (D) Quantification of CaV1.2-expressing h-MSCs (n = 13) and AML-MSCs (n = 14) in 2D culture, analyzed by flow cytometry. (E) Relative mRNA expression of CaV1.2 (CACNA1C) in h-MSCs (n = 12) and AML-MSCs (n = 16) in 2D culture, measured by RQ-polymerase chain reaction. (F) Intracellular calcium increases in MSCs in 2D culture loaded with calcium indicator Fluo-4 AM probe and stimulated with KCl (n = 2, arbitrary unit [AU]). (G) Intracellular calcium increases in MSCs in 2D culture loaded with calcium indicator Fluo-4 AM probe after CaCl₂ addition (5 mM) to culture medium on h-MSCs or AML-MSCs under lercanidipine or vehicle (DMSO) treatment (n = 3). (H) Cell proliferation (by ATP assay) and viability (by Annexin V/PI staining) of AML-MSCs in 2D culture up to 9 days after lercanidipine treatment. Dotted lines represent cell proliferation and viability after washout (w/o) at 24 hours from lercanidipine treatment (n = 5). *P < .05, **P < .01, ***P < .001, ****P < .0001. RQ, real quantitative.