Fig. 3
ALPL deficiency promoted the internalization of L-type Ca2+ channels in BMSCs. a, b Representative images of confocal laser scanning microscopy showing the membrane location of CaV1.2 and CaV1.3 (green) in alpl+/− BMSCs transfected with DN-Dyn1. The plasma membrane was stained with the marker CellMask™ Deep Red Plasma Membrane Stain (red) (a). Quantification of the membrane florescence was performed with NIH ImageJ (b). Scale bar, 10 μm. c alpl+/− BMSCs transfected with DN-Dyn1 showed upregulation of CaV1.2 and CaV1.3 membrane expression and almost no change in the cytoplasmic expression of CaV1.2 and CaV1.3, as assessed by western blot. β-actin was used as a protein loading control. d 10-min time-lapse confocal laser scanning microscopy images of WT, alpl+/−, DN-Dyn1/alpl+/−, and Lenti-alp/alpl+/− BMSCs containing DsRed-CaV1.2. Scale bar, 10 μm. e Ca2+ imaging showed elevated Ca2+ influx of cultured alpl+/− BMSCs transfected with DN-Dyn1 after stimulation with 30 mmol·L−1 KCl for 3 min (n = 10). alpl+/− BMSCs showed a pronounced decrease in DsRed-CaV1.2 at the membrane (Dio-labeled ROI, n = 10) (f). g Quantification of the florescence in the ROI during the time-course lapse at 0 s, 300 s, and 600 s. h–k Representative images show the colocalization of DsRed-CaV1.2 with the Dio-labeled cell membrane of BMSCs. WT, DN-Dyn1/alpl+/−, and Lenti-alp/alpl+/− BMSCs had colocalized regions at 0 s and 570 s (h, j, k). However, alpl+/− BMSCs showed no colocalization at 0 s and 570 s (i). 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
