Fig 2: Immunofluorocytochemical analysis showing formation of tissue-nonspecific alkaline phosphatase (TNSALP) by aesculetin. MC3T3-E1 cells were cultured in differentiation media in the absence or presence of 1–10 µM aesculetin for 21 days. The matrix vesicle formation was confirmed by FITC-green staining of TNSALP on the top of differentiated MC3T3-E1 cells (n = 3). Nuclear staining of these cells was carried out with 4', 6-diamidino-2-phenylindole (DAPI, blue), and visualized under light microscopy. Scale bar = 50 µm.

Fig 3: The identification of vitamin K-dependent NCPs in rat and turkey and a comparison of MGP abundance in rat (a) and turkey (b). The identification of OCN in rat and turkey is validated by Western blotting (c). Western blot results of tissue-nonspecific alkaline phosphatase (TNAP) in rat bone and tail tendon (d). B: rat bone; T: rat tail tendon; MT: mineralizing TLT; NT: not-yet-mineralized TLT; NMT: never-mineralizing TLT. G, G1, G2, and E: extraction steps as defined in the text and in Fig. 1. 1, 2: Specimens 1 and 2.

Fig 4: Altered glycan processing affects the function of MSC derived osteoblasts. (A) Alizarin Red staining of WT or Cog4KD Y101 hTERT-MSC cells following a 21 day differentiation experiment in osteogenic medium. Quantification of the eluted Alizarin Red stain is shown in the graph below the image. Results are mean±s.d. (n=6). (B) ALP and von Kossa staining of Y101 cells or Y101 cells stably expressing a Cog4-specific shRNA (shRNA2) following incubation in osteogenic medium with the indicated inhibitors for 21 days. NB-DNJ, N-butyldeoxynojirimycin; BG, benzyl-O-GalNAc. Scale bar: 100 µm. (C) Real-time qPCR analysis of mRNA expression levels of indicated genes in the Y101 WT or shRNA KD cells. Medium was supplemented with the indicated inhibitors 2 days prior to the start of and until the end of the differentiation experiments. Averages of triplicate measurements were normalized to the day zero control. Data represent the mean of the mean of three technical repeats each for two independent biological replicates (each normalized independently to its own control) with s.e.m. shown. The bar positions marked with ‘X’ are from the 21 day shRNA2 samples that peeled off. All qPCR experiments in Figs 2C and 3B were performed in parallel, and used the same untreated WT as control. This control is replicated in all three rows of 2C as well as 3B for clarity. *P<0.05, **P<0.01, ***P<0.001 (compared to the control samples are marked with asterisks); all other changes are non-significant.

Fig 5: Schematic diagram showing the effects of aesculetin on osteoblastogenesis and collagen mineralization. The arrows indicate activation by aesculetin. ALP, alkaline phosphatase; BMP-2, bone morphogenetic protein-2; BSP, bone sialoprotein; OPG, osteoprotegerin; Runx2, Runt-related transcription factor 2; TNSALP, tissue-nonspecific alkaline phosphatase.