Figure 6: PKD1 contributed to osteoblastic development.

(a) ALP staining and ALP-positive cell numbers of MC3T3-E1 and MG63 cells treated with 10 μM of CRT0066101(CRT) for 24 hours, positive numbers represent the mean ± SD of 3 wells (*p < 0.05). (b) MC3T3-E1 was transfected with siRNA of PKD1(si-PKD1) and negative control (si-CTL) for 48 hours. Osteoblastic markers (OPN and Runx2) and PKD1 expression were analyzed by Western blotting. The density of proteins bands was scanned and the values were normalized to control. (c) 0 day or 3 days differentiating MC3T3-E1 was treated with DMSO or 10 μM of CRT0066101 for 24 hours and then subjected to Western Blotting. (d) After days 3, 5, 8 differentiating, MC3T3-E1 cells were treated with CRT0066101 for 24 hours. Expression of PKD1 phosphorylation at ser916 and osteoblast regulators (OPN, Runx2 and OSX) were analyzed by Western Blotting. (e) Primary calvarial preosteoblast on the day of cell plating (day 0), on the 8^th and 12^th day of osteogenic differentiation were analyzed by Western blotting to detect expression of osteoblast markers Runx2, and OPN in WT or KO mice (n = 3). All Band densities were normalized to α-tublin content within each sample, and normalized to Day 0 at the far left WT group, control = 1.00. (f,g) PKD1, ser916 of PKD1phosphorylation, proliferative markers PCNA and Cyclin D1 were analyzed at the 0, 3^rd, 5^th and 7^th day growth in the MC3T3-E1 and MG63 cells. Representative Western Blots were shown. Uncropped western blot images corresponding to Fig. 6(b,c,d). Figure 6(e,f and g) were shown in supplementary information file.