Fig 2: MiR-185 silencing promotes the osteogenic function of mesenchymal stem cells (MSCs).MSCs were derived from the bone marrow of wild-type (WT) or knockout (KO) mice, and induced with osteoblast induction medium (OIM) for osteogenic differentiation. a Alkaline phosphatase (ALP) staining in WT or KO MSCs after osteoblast induction for 7 days. Scale bar = 500 µm. b ALP activity determination in MSCs after osteogenic induction for 7 days. c Alizarin Red S Staining in MSCs after osteoblast induction for 21 days. Scale bar = 500 µm. d Matrix mineralization was quantified in MSCs after induction for 21 days. e Real-time PCR showed the messenger RNA (mRNA) expression levels of osterix (Osx), collagen type 1a 1 (Col1a1), and osteocalcin (OC) in MSCs after induction with OIM for 7 days. f The expression of osteoblast marker genes in MSCs cultured for 3 days in OIM was indicated by western blot. Data were shown as mean ± S.D (*P < 0.05, **P < 0.01, ***P < 0.001)

Fig 3: PKD1 impaired osteoblast differentiation through JAK1/STAT3 and p38 signaling pathway.(a) After days 3, 5 differentiating, MC3T3-E1 cells were treated with CRT0066101 or vehicle for 24 hours and then underwent immunoblotting to detect PKD1, PKD1 phosphorylation activity, JAK1, STAT3 and phosphorylation (ser727). Photoshop analyzed the density of bands on western blot. For each marker band, values were normalized to control and the density of control was set at 1. (b) Western Blot analysis of differentiating primary calvarial preosteoblasts showed decreased expression of p38,P-p38,STAT3,P-STAT3-ser727, y705 and PCNA in KO cells (0, 12th day). n = 3, WT or KO mouse. (c) MC3T3-EI cells were transfected with si-PKD1 and negative control (si-CTL), pcDNA PKD1 and empty vector plasmid for 60 hours to analyze osteoblast regulators (Runx2, OPN,OSX and OCN) and relative pathway markers (STAT3 and p38). (d) MC3T3-EI cells were transfected with si-STAT3 and negative control (si-CTL) for 60 hours. Uncropped western blot images corresponding to Fig. 7(a,b,c and d) were shown in Supplementary Information file.

Fig 4: 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 8th and 12th 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 a-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, 3rd, 5th and 7th 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.

Fig 5: PLGA scaffold composite of BMP-9 and P-15 peptide hydrogel promoted ALP, COL-1, OCN, RUNX2, and Sp7 expression in bone tissue. Compared with the control group, *P < 0.05. Compared with the model group, #P < 0.05.