Fig. 1

ALPL deficiency caused decreased membrane expression of L-type Ca²⁺ channels in BMSCs. a Ca²⁺ imaging showed decreased Ca²⁺ influx in cultured alpl^+/− BMSCs and WT BMSCs transfected with shALP (shALP/WT) after they were stimulated with 30 mmol·L⁻¹ KCl for 3 min (n = 10). b No KCl-induced Ca²⁺ influx was detected in cultured WT, alpl^+/−, and shALP/WT BMSCs treated with 10 mmol·L⁻¹ EGTA for 3 min (n = 10). c ALPL overexpression was mediated by a lentivirus in alpl^+/− (Lenti-alp/alpl^+/−) BMSCs and resulted in an elevated Ca²⁺ influx following stimulation with 30 mmol·L⁻¹ KCl for 3 min (n = 10). d, e The expression of Ca_V1.1, Ca_V1.2, and Ca_V1.3 was assessed. alpl^+/− BMSCs showed decreases in total cell expression (d) and membrane expression of Ca_V1.2 and Ca_V1.3 (e) and no significant change in the levels of cytoplasmic Ca_V1.2 and Ca_V1.3 (e). Total Ca_V1.1 protein expression was not changed (d), and the expression of membrane and cytoplasmic Ca_V1.1 was not altered in alpl^+/− BMSCs (e). f Cell-surface biotinylation assay. Left two lanes: western blot for Ca_V1.2 and Ca_V1.3 following neutravidin pull down from WT and alpl^+/− BMSCs; right two lanes: input, not biotinylated cells. g Lenti-alp/alpl^+/− BMSCs showed elevated membrane expression of ALP, Ca_V1.2, and Ca_V1.3. h, i Representative images of confocal laser scanning microscopy showing the membrane location of Ca_V1.2 and Ca_V1.3 (green) in WT and Lenti-alp/alpl^+/− BMSCs. The plasma membrane was stained with the marker CellMask™ Deep Red Plasma Membrane Stain (red) (h). Quantification of the membrane florescence was performed with NIH ImageJ (i). Scale bar, 10 μm. 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