Fig. 7: Alpl also controls the differentiation and senescence of human MSCs via ATP-mediated inactivation of the AMPKα pathway.

a SA-β-gal staining and Ki67, γH2AX and LAP2β immunostaining of third-passage MSCs from normal controls and HPP patients. Quantification of Ki67⁺, γH2AX⁺ and LAP2β⁺ is indicated in the right panel. Scale bars: 50 μm. b Expression levels of ageing-specific genes in normal and HPP yiMSCs were examined by western blotting. Scale bars, 50 μm. c Expression levels of CD73 and CD39 in normal and HPP MSCs were examined by western blotting. d Extracellular ATP concentrations in normal and HPP MSC medium were examined by a regular ATP concentration assay. e Intracellular radioactivity was examined after a 1-h treatment with ATP-γ-³²P in different lentiviral vector transduction groups. f Intracellular ATP concentrations were assayed 48 h after the transduction of different lentiviral vectors. g Western blotting analysis of p-AMPKα expression in normal control and the ALPL shRNA, HPP control and pLenti-ALPL groups. h Expression levels of p16 and p53 were assayed 48 h after the transduction of different lentiviral vectors. i HPP MSCs overexpressing ALPL or treatment with 0.1 mmol·L⁻¹ metformin, Alizarin Red staining and quantification of mineralized nodules were performed on day 28 after osteogenic induction (OS). Expression levels of Runx2 and OCN were examined by western blotting on day 7 after induction. j Oil Red O staining and quantification of fat depots were performed on day 14 after the adipogenic induction (AD). PPAR-γ expression was examined on day 7 after induction by western blotting. Scale bars, 100 μm. (N) Normal control n = 5, HPP (hypophosphatasia patient) n = 2. The data are presented as the means ± s.d. of each independent experiment performed in triplicate. *P < 0.05, **P < 0.01. e–f, i–j One-way analysis of variance (ANOVA). a, d Unpaired two-tailed Student’s t-test.