Fig 1: Determination of ALP activity, and gene expressions of ALP and Osteocalcin. Kcp accelerates the effect of BMP-2 to induce differentiation into osteoblasts. (A(i)) Measurement of ALP activity. (A(ii)) ALP activity staining. BMP-2 (300 ng·mL-1) and Kcp (400 ng·mL-1) were added to C2C12 cells derived from mouse myoblasts. After 72 h of incubation at 37 °C, cells were subjected to destruction with an ultrasonic destructor in order to measure ALP activity within the cells by determining absorbance. Relative values for each group were calculated based on a control group value of 1. (B) Gene expression of ALP and Osteocalcin as marker genes for osteoblast differentiation. C2C12 cells derived from mouse myoblasts were used with BMP-2 and Kcp added. The level of ALP gene expression was analyzed using a quantitative PCR method with a TaqMan probe. ALP expression level was corrected using GAPDH expression level as the intrinsic control. The relative expression level of each group was calculated using an ALP/GAPDH value of 1 for the group without BMP-2 and Kcp added (control). Results are shown as the mean ± SD of three samples. **P < 0.01 and *P < 0.05, Student's t-test. Scale bars = 200 µm.
Fig 2: Determination of ALP activity with various concentrations of reagents. (A) C2C12 cells with a stable concentration of BMP-2 (300 ng·mL-1) and various concentrations of Kcp (1–1000 ng·mL-1). (B) ALP activity staining. Scale bars = 1000 µm. (C) C2C12 cells with a stable concentration of Kcp and various concentrations of BMP-2 (3–1000 ng·mL-1). After 72 h of incubation at 37 °C, cells were subjected to destruction with an ultrasonic destructor; then, ALP activity within the cells was determined by measuring absorbance. (D) ALP activity staining. Scale bars = 200 µm. Results are shown as the mean ± SD of three samples. **P < 0.01 and *P < 0.05, Student's t-test.
Fig 3: Alizarin red staining for analyzing calcification of osteoblasts. C2C12 cells derived from mouse myoblasts were used to examine the effects of BMP-2 and Kcp. The time course for differentiation and calcification of the osteoblasts was 14 days. Differentiation medium containing ascorbic acid, ß-glycerophosphate (10 mm), and dexamethasone was used, with that changed every 3 days. (A) Determination of calcification based on absorbance of Alizarin red. (B) Alizarin red staining. Dark red areas indicate osteoblast calcification. Results are shown as the mean ± SD of three samples. **P < 0.01, Student's t-test. Scale bars = 200 µm.
Fig 4: Western blotting analysis to detect activation of Smad with phosphorylation. (A) Band showing phosphorylated Smad 1/5 protein with application of both BMP-2 and Kcp indicates an apparently increase as compared to without Kcp application. There were no differences regarding the protein amounts of Smad1, Smad5, and actin among the groups. (B) Graph showing relative values of adjusted volume of protein bands for pSmad1/5. The adjusted volume of pSmad1/5 was determined based on the adjusted volume of actin. Relative values for each group were determined using a value of 1 for the control group. (C) Cell signaling pathway showing activity of BMP-2 and Kcp to phosphorylate Smad.
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