Fig. 3: The osteocytic Ca²⁺ oscillatory response to mechanical loading is compromised in T2D both in vitro and in situ.

a The experimental protocol of real-time osteocyte Ca²⁺ imaging in the tibiae of male T2D mice in situ subjected to cyclic compressive loading using the synchronized cyclic loading/confocal imaging technique. b Representative intracellular Ca²⁺ profiles of osteocytes in situ in diabetic and non-diabetic tibiae in response to cyclic mechanical loading, and the corresponding statistical results, including the percentage of responsive cells, the number of Ca²⁺ spikes, the average Ca²⁺ intensity, and the relaxation time of Ca²⁺ spikes. c The experimental protocol of real-time intracellular Ca²⁺ imaging in MLO-Y4 osteocytic cells and primary osteoblasts in vitro exposed to the high-glucose and high-fat (HGHF) condition for 48 h in response to subsequent fluid shear stress (FSS) stimulation. d Comparison of intracellular Ca²⁺ dynamics of MLO-Y4 osteocytic cells in vitro exposed to normal medium, mannitol-treated medium (MAN, osmotic pressure-matched control), and HGHF-treated medium in response to subsequent steady FSS stimulation (2 Pa). e Comparison of intracellular Ca²⁺ dynamics of normal, mannitol-treated, and HGHF-treated primary osteoblasts in vitro under steady FSS stimulation (2 Pa). Graphs represent mean ± SD (n = 120 cells per group). *P < 0.05 and ***P < 0.001 by one-way ANOVA with Bonferroni’s post test. Specific P values are provided in the Source Data file. Scale bars: b 20 μm; d, e 30 μm.