Figure 4
Combination of SCX overexpression and mechanical stress synergistically induces tendon differentiation through differential regulation of BMP.
(a) Endogenous SCX expression of cells treated with BMP2 or Noggin after 12 h to 3 days ( n = 3, *, P<0.05 noggin effects). (b) The morphology of the cells treated with or without noggin. (c) Tenocyte gene expression of engineered tendon with or without noggin treatment after day 3 and day 7 (n = 2, fold over control group without noggin). (d) hESC-MSC, Hela and C3H10T1/2 cells were transiently transfected with BMP2 promoter reporter vector alone, or with increasing amounts of expression vectors encoding scleraxis after 24 h. Luciferase expression levels were normalized to co-transfected rentilia vector. (n> = 3). *, P<0.05 compared to control. (e) Western blot analysis of phosphorylation of Smad1/5/8 on control and SCX overexpressing cells treated with BMPs 1 hour. Beta-actin and Smad1-Smad5-Smad8 served as loading control. Representative results from 3 independent experiments. (f) Immunostaining of Runx2 in control, SCX overexpressing and noggin treated cells after 3 days. Arrows are pointing to cells which are shown at high magnification as inserts at the bottom left corners. (g) Immunostaining of Eya2 in control, SCX overexpressing and noggin treated cells after 3 days. Arrows are pointing to cells which are shown at high magnification as inserts at the bottom left corners. Scale bar, 50 um (f, g).
