Alkaline pHi and MCT4 upregulation is common in AML. (A) Ex vivo pHi of mouse hematopoietic malignancies and normal blood cells examined using SNARF-1 by FACS (n = 3-9). Statistical comparisons between different groups were performed (progenitors/differentiated cells vs long-term hematopoietic stem cells [LT-HSCs]; AML LICs vs LT-HSCs; AML vs LKS). (B) Ex vivo pHi of human AML cell lines and CB CD34⁺ and normal BM mononuclear cells (BMMCs) examined using SNARF-1 by FACS (n = 3). Statistical comparisons between AML cell lines and CB were performed. (C) Ex vivo pHi of primary human AML (n = 12) and primary AML CD34⁺CD38⁻ (n = 7) with CB HSC1 (n = 3), CB HSC2 (n = 3), CB (n = 7), and BMMCs (n = 7) examined using SNARF-1 by FACS. (D) In vivo pHi of 100 normal blood cells and 100 MLL-AF9 AML cells (from 3 mice) imaged by multiphoton fluorescence microscope were analyzed by ImageJ. pHi was determined based on the calibrated standard cell (supplemental Figure 1C). (E) Representative images examined by in vivo multiphoton fluorescence microscope showing that mouse MLL-AF9 AML was more greenish in color, whereas normal blood cells were more reddish in mouse calvarial BM cavity. (F) Quantitative PCR (qPCR) analysis of transcriptional expression of MCT4 in mouse LT-HSCs (n = 6), short-term HSCs (ST-HSCs; n = 6), MLL-AF9 AML LICs (n = 3), HoxA9-Meis1 AML LICs (n = 3), and FLT3^ITD/TET2^−/− AML LICs (n = 4). (G) qPCR analysis of transcriptional expression of MCT4 in human CB HSC1 (n = 3), CB HSC2 (n = 3), CB multipotential progenitors (MPPs; n = 3), CB progenitors (n = 3), primary AML CD34⁺CD38⁻ (n = 6), and primary bulk AML (n = 6). (H) Western blot of MCT4 protein in primary human AML (n = 16) and CB CD34⁺ (n = 3). (I) Western blot of MCT4 protein in the subpopulation of different pHi of primary human AML (n = 3; number showed the intensity of MCT4 in pH^high (H) cells normalized by that in pH^low (L) cells]. *P < .05, **P < .01, ***P < .001. ns, not significant.