MDH1 enhances the malate-aspartate NADH shuttle and decreases NADH/NAD⁺ level in SoNar-low FL-HSCs. (A-B) Gene ontology (GO; biological process; A) and Kyoto Encyclopedia of Genes and Genomes (KEGG; pathway; B) analyses of the microarray data of CD45⁺ SoNar-high vs -low FL hematopoietic cells. Candidate changes (downregulated genes) are highlighted in red. (C) Potential candidates related to glycolysis, monocarboxylate transporter, malate-aspartate shuttle, oxidative phosphorylation, pentose phosphate pathway (PPP), and glutamine and serine metabolism were evaluated in CD45⁺ SoNar-high vs -low FL hematopoietic cells by quantitative RT-PCR (n = 3). (D) Protein levels of MDH1 in CD45⁺ SoNar-high and -low FL hematopoietic cells were measured by immunoblotting (n = 3). Ratio of MDH1/actin was quantified and normalized against SoNar-high#1 cells. (E) Mdh1 expression levels in total CD45⁺ FL hematopoietic cells, FL-HSCs, SoNar-high FL-HSCs, and SoNar-low FL-HSCs were determined by quantitative RT-PCR (n = 3). (F) Representative images for the ratios of SoNar fluorescence of Mdh1-knockdown and scrambled SoNar 32D cells. (G) Quantification of the ratios of SoNar in panel F. A total of 30 SoNar 32D cells were analyzed (n = 3). (H) Quantification of the ratios of SoNar fluorescence in SoNar 32D cells at indicated time points upon AOA incubation. A total of 22 to 25 SoNar 32D cells were analyzed (n = 3). (I) Mdh1 was silenced in CD45⁺ FL hematopoietic cells by short-hairpin RNA (shRNA) targeting Mdh1 (shMdh1#2 or shMdh1#3) followed by transplantation into recipients. The repopulation was analyzed at 4, 8, and 16 weeks posttransplantation (n = 5). (J-K) Multilineage contribution of donor cells in the recipients at 16 weeks posttransplantation (J; n = 5). MDH1 levels were measured in BM cells of recipients by immunoblotting (K). Ratio of MDH1/actin was quantified and normalized against scrambled cells. (L) CD45⁺ SoNar-high and -low FL hematopoietic cells were treated with AOA for 24 hours, followed by transplantation into recipients (n = 5). Repopulation was analyzed at 4, 8, and 16 weeks posttransplantation. Scale bar, 10 μm. Data are represented as mean ± standard error of the mean. One-way analysis of variance (ANOVA) with Tukey’s multiple comparison test (C,G) and 2-way ANOVA with Sidak’s multiple comparison test (I-J,L) were used for the comparison. See also supplemental Figure 6. *P < .05, **P < .01, ***P < .001. ER, endoplasmic reticulum.