Fig. 5: Increasing SERCA2 expression in T2D skeletons enhances biological activities and Ca²⁺ oscillatory responses to mechanical loading in osteocytes, although not in osteoblasts.

a Real-time intracellular Ca²⁺ signaling of osteocytes in situ in the tibiae of male KK-Ay mice treated with the SERCA2 agonist istaroxime (ISTA, 1 μM) in response to axial cyclic compressive loading. b Intracellular Ca²⁺ dynamics of osteocytes in situ in the tibiae of high-fat diet/streptozotocin-treated (HFD + STZ) male mice incubated with ISTA in response to cyclic compressive loading. c Intracellular Ca²⁺ dynamics of MLO-Y4 osteocytic cells in vitro treated with high glucose and high-fat (HGHF) and the SERCA2 agonist ISTA (1 μM) in response to steady FSS stimulation (2 Pa). d Intracellular Ca²⁺ dynamics of primary osteoblasts in vitro treated with HGHF and ISTA in response to steady FSS stimulation. e The Annexin V-FITC/PI apoptosis assays in MLO-Y4 cells treated with HGHF and ISTA in response to FSS stimulation (2 Pa). f Western blotting assays in MLO-Y4 cells treated with HGHF and ISTA in response to FSS, including β-catenin, OPG, RANKL, and DKK1. g Western blotting assays in primary osteoblasts treated with HGHF and ISTA in response to FSS, including Col1a1, Osx, and Runx2. h–j The ALP activity, ALP staining, and Alizarin red staining in primary osteoblasts treated with HGHF and ISTA in response to FSS stimulation. Graphs represent mean ± SD (a–d n = 120 cells per group; e–j n = 6 biologically independent replicates). a–d ***P < 0.001 by two-tailed Student’s t test. e–j *P < 0.05 and ***P < 0.001 by two-way ANOVA with Bonferroni’s post test. Specific P values are provided in the Source Data file. Scale bars: h, j 50 μm.