Fig. 4. Supplementation of citrate, glutamine, and magnesium synergistically enhanced energy metabolism.

(A) Intracellular ATP level on day 7 of hBM-MSC osteogenic differentiation with various glutamine concentrations. Data are average $\pm$ SD. One-way ANOVA with Tukey’s multiple comparisons test, *P < 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.0001. n = 3 to 6. (B) Intracellular ATP level on day 7 of hBM-MSC osteogenic differentiation with various magnesium concentrations. Data are average $\pm$ SD. One-way ANOVA with Tukey’s multiple comparisons test, *P < 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.0001. n = 4. (C) Intracellular ATP level on day 7 of hBM-MSC osteogenic differentiation with control, CA, CA + Mg, CA + Gln, or CA + Gln + Mg. Data are average $\pm$ SD. One-way ANOVA with Tukey’s multiple comparisons test, *P < 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.0001. n = 3 to 5. (D) OCR profile plot, (E) basal respiration, maximal respiration, ATP production, and proton leak at day 7 of hBM-MSCs ostegeogenic differentiation with control, CA, or CA + Gln + Mg. Data are average $\pm$ SD. One-way ANOVA with Tukey’s multiple comparisons test, *P < 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.0001. n = 4. (F) Representative images of mitochondria membrane potential on day 7 of hBM-MSCs osteogenic differentiation with control, CA, or CA + Gln + Mg. Scale bar, 50 μm. (G) Intracellular levels of metabolites in TCA cycle on day 3 of hBM-MSCs osteogenic differentiation with control, CA, or CA + Gln + Mg. (H) Heatmap illustrating the intensity value distribution of 138 differential metabolites (DMs) across groups with CA, Gln, Mg, or CA + Gln + Mg. The significantly enriched KEGG pathways and their associated key metabolites from each cluster were displayed on the right side of the heatmap. Rot/AA, rotenone/antimycin A; FCCP, carbonyl cyanide p-trifluoromethoxyphenylhydrazone.