|
|
|
|
|
|
|
|
|||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | |||||
|
Fig. 5. Endofin regulates expression of BMP downstream genes and mineralization in human MSCs. (A) Transcriptional response assay. C2C12 cells stably expressing GFP (control), or endofin or its mutants were transfected with BMP signaling reporter construct 9XSBE-luc. Transfected cells were incubated in the presence or absence of BMP2 (200 ng/ml). Luciferase activity was normalized and plotted as the mean ± s.d. of triplicates from a representative experiment. *P<0.05 compared with the second group. (B) Alkaline phosphatase activity assay. C2C12 cells stably expressing GFP (control) or endofin or its mutant PBD(F872A) were cultured in 24-well plates treated with or without BMP2 and harvested at day 5 for alkaline phosphatase activity assay. Relative alkaline phosphatase activity was normalized and plotted as the mean ± s.d. of triplicates from a representative experiment. *P<0.05 compared with second group. (C) Alkaline phosphatase staining. C2C12 were cultured and treated as in alkaline phosphatase activity assay and harvested at day 3. After fixation with 4% paraformaldehyde, cells were subjected to alkaline phosphatase staining. Bar, 20 µm. (D) Overexpression of mutated endofin with disrupted PP1c-binding domain enhanced mineralization activity of human MSCs (von Kossa Assay). Human MSCs were cultured in Dulbecco's Modified Eagle's Medium: low glucose, 1x penicillin-streptomycin, 10% fetal bovine serum (BioWhittaker, MSC serum), 10 mM 
-glycerol phosphate, 50 µM Ascorbic acid 2-phosphate (AsAP) and 200 ng/ml BMP2. Mineralization assay was performed at day 24. After silver nitrate was added, a calcium deposit was visible as a black structure or focal dot. i, GFP; ii, GFP+BMP2; iii, Endofin+BMP2; iv, Endofin-PBD(F872A)+BMP2. Bar, 30 µm.
|
