Fig. 2

ALPL-maintained MSC osteogenic/adipogenic lineage differentiation ability via the L-type Ca²⁺ channel. a Ca²⁺ imaging showed elevated Ca²⁺ influx in alpl^+/− BMSCs transfected with oeCa_V1.2 or oeCa_V1.3 following stimulation with 30 mmol·L⁻¹ KCl for 3 min (n = 10). b, c Representative images of confocal laser scanning microscopy showing the membrane location of Ca_V1.2 and Ca_V1.3 (green) in alpl^+/− BMSCs transfected with oeCa_V1.2 or oeCa_V1.3. The plasma membrane was stained with the marker CellMask™ Deep Red Plasma Membrane Stain (red) (b). Quantification of the membrane florescence was performed with NIH ImageJ (c). Scale bar, 10 μm. Alizarin red staining showed that alpl^+/− BMSCs transfected with oeCa_V1.2 or oeCa_V1.3 had an increased capacity to form mineralized nodules when cultured under osteoinductive conditions (d) and they exhibited an upregulation of the osteogenic-related proteins RUNX2 and Sp7 (e). oeCa_V1.2- or oeCa_V1.3-treated alpl^+/− BMSCs showed a decreased number of oil red O-positive adipocytes when cultured under adipo-inductive conditions (f) and there was a downregulation of the adipogenic-related proteins PPARγ2 and LPL, as assessed by western blot (g). h Alizarin red staining showed that alpl^+/− BMSCs transfected with siCa_V1.2 or siCa_V1.3 had a decreased capacity to form mineralized nodules when cultured under osteoinductive conditions. i Western blot analysis showed that BMSCs transfected with siCa_V1.2 or siCa_V1.3 expressed decreased levels of the osteogenic-related proteins RUNX2 and Sp7. β-actin was used as a protein loading control. BMSCs transfected with siCa_V1.2 or siCa_V1.3 showed an increased number of oil red O-positive adipocytes when cultured under adipo-inductive conditions (j) and upregulation of the adipogenic-related proteins PPARγ2 and LPL, as assessed by western blot (k). The representative results from three independent experiments are shown. Error bars represent the s.d. from the mean values. *P < 0.05; **P < 0.01; ***P < 0.001