Figure 4. Acod1-deficiency promotes switch towards glycolysis in a Hif1α-dependent manner. (A) Time course analysis of glycolytic and mitochondrial activity in wild-type (WT) cells throughout osteoclast differentiation, determined by mitochondrial and glycolytic stress test assays on days 0, 1, 2 and 3 of osteoclast cell culture and quantification of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) as a measure for basal respiration and glycolysis, respectively (n=6–13). (B) Quantification of the percentage of glycolytic versus mitochondrial adenosine triphosphate (ATP) production in osteoclasts (day 3 of cell culture) from WT and aconitate decarboxylase 1-deficient (Acod1^−/−) mice, determined by ATP rate assay (n=12–15). (C) OCR profile plot and (D) mitochondrial function parameters in osteoclasts (day 3 of cell culture) from WT and Acod1^−/− mice, determined by mitochondrial stress test assay (n=25–29). (E) ECAR profile plot and (F) glycolytic activity parameters in WT and Acod1^−/− osteoclasts (day 3 of cell culture), determined by glycolytic stress test assay (n=12–16). (G) Representative images of immunofluorescent staining for hypoxia-inducible factor (Hif)-1α (yellow), F-actin (purple) and DAPI (blue) and (H) fold change quantification of the Hif1α/DAPI mean fluorescence intensity (MFI) in osteoclasts (day 3 of cell culture) from Acod1^−/− as compared with WT mice (n=5). Scale bar: 50 µm. (I) Flow cytometry analysis of Hif1α expression in CD45⁺CD11b⁺Ly6C^hiCD115⁺ osteoclast precursors (OCPs) in the synovial ankle tissue of WT and Acod1^−/− mice with serum-induced arthritis (SIA), depicted as fold changes of the MFI in Acod1^−/− as compared with WT cells (n=5). (J) Representative images and (K) quantification of tartrate-resistant acid phosphatase (TRAP)-stained polynucleated osteoclasts (≥5 nuclei) from Acod1^−/− mice that were subjected to CRISPR/Cas9-mediated deletion of Hif1a gene or from WT and Acod1^−/− mice that were treated with a non-targeting control construct on day 0 of osteoclast cell culture (n=5). Scale bar: 100 µm. Data are shown as mean±SEM. Symbols represent individual mice. P values were determined by two-tailed Student’s t-test for single comparisons and one-way ANOVA or two-way ANOVA for multiple comparisons. ANOVA, analysis of variance.