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. 2025 Jul 23;11(30):eady2862. doi: 10.1126/sciadv.ady2862

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Copyright © 2025 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).

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Fig. 5. — (A) Protein expression of p70-S6K-pT389, AMPKα-pT172, and AKT-pT308 at different days of hBM-MSCs osteogenic differentiation. (B) Protein expression of p70-S6K-pT389 and AMPKα-pT172 on day 3 of hBM-MSCs osteogenic differentiation with different treatments. (C) Protein expression of p70-S6K-pT389 on day 3 of hBM-MSCs osteogenic differentiation with Torin1 followed by different treatments. (D) Principal components analysis (PCA) of metabolomic data of hBM-MSCs osteogenic differentiation with different treatment conditions (left), and the intensity value distribution of ATP in the samples (right). The arrows denote the direction of three effects after different treatments. (E) ALP activity on day 7 of hBM-MSCs osteogenic differentiation without (NT) or with STO609 followed by without (control) or with CA + Gln + Mg treatment. Data are average $\pm$ SD. Two-tailed unpaired t test, *P < 0.05, **P < 0.005, ***P < 0.0005, and ****P < 0.0001. n = 4. (F) Protein expression of AMPKα-pT172 on day 3 of hBM-MSCs osteogenic differentiation with STO609, followed by different treatments. (G) Protein expression of p70-S6K-pT389 on day 3 of hBM-MSCs osteogenic differentiation with or without STO609, followed by different treatments. (H) Protein expression of p70-S6K-pT389 on day 3 of hBM-MSCs osteogenic differentiation with MK2206 followed by different treatments. (I) ALP activity on day 7 of hBM-MSCs osteogenic differentiation without (NT) or with MK2206 followed by without (control) or with CA + Gln + Mg treatment. Data are average $\pm$ SD. Two-tailed unpaired t test, *P < 0.05. n = 4. n.s., not significant. (J) Protein expression of AKT-pT308 on day 3 of hBM-MSCs osteogenic differentiation with different treatments. (K) Expression of core genes in the CaMKK2 pathway (CaMKK2), AMPK pathway (PRKAA1), AKT pathway (AKT1), and mTOR pathway (MTOR, RPTOR, and RPS8KB1) within the human osteogenic population. (L) Schematic representation of the mTOR-AMPK network with citrate, glutamine, and magnesium supplementations. Figure created in BioRender [https://BioRender.com/a90y633; Hui Xu (2025)].