AML infiltration inhibits osteogenesis. (A) Alizarin red staining to detect calcium deposition after 14 days of induction of mineralization in HuMSPCs in the presence of PBMNCs from healthy donors (control) or different AML cell lines. Left: low-magnification image of a 48-well plate; right: images (4× objective) showing calcium deposition. (B) Colorimetric detection of alizarin red staining using absorbance at 450 nm to quantify calcium deposition in HuMSPCs after 14 days of induction of mineralization in the presence of healthy PBMNCs as controls or different AML cell lines (representative of 9 independent experiments). (C) Colorimetric detection of alizarin red staining using absorbance at 450 nm to quantify calcium deposition in SaOS2 cells after 7 days of induction of mineralization in the presence of controls or different AML cell lines (representative of 6 independent experiments). (D) Images (4× objective) of 96-well plate and colorimetric detection of alizarin red staining using absorbance at 450 nm to quantify calcium deposition in HuMSPCs after 14 days of induction of mineralization in the presence of controls or primary AML cells (3 replicates per sample). (E) Image (4× objective) and colorimetric detection of alizarin red staining using absorbance at 450 nm to quantify calcium deposition in HuMSPCs after 21 days of induction of mineralization in the presence of different AML cell lines or corresponding conditioned medium (representative of 3 independent experiments). Data are shown as mean ± SEM. *P < .05; **P < .01; ***P < .001; ****P < .0001 (determined by unpaired Student t test).