Figure 2: The effects of CKIP-1 knockdown and Smad1 overexpression on osteogenic activity and Smad-dependent BMP signaling in glucocorticoid-treated MC3T3-E1 cells in vitro.

(a) The mRNA expression levels of CKIP-1 and osteogenic marker genes (ALP, COL1, OPN, BSP and OCN) in GT and Control groups at day 3, 5 and 7, respectively. (b) The amount of deposited calcium in GT and Control groups (left) at day 7, respectively. (c) Quantification of pSmad1/5 protein levels in GT and Control groups at day 3, 5 and 7, respectively. (d) Representative electrophoretic bands on the protein expression of CKIP-1, pSmad1/5, Smad1/5 and Smurf1 in GT and Control groups at day 3, 5 and 7, respectively. (e) The mRNA expression levels of CKIP-1 and osteogenic marker genes (ALP, COL1, OPN, BSP and OCN) in RNAi, VC and NC groups at day 3, 5 and 7, respectively. (f) The amount of deposited calcium in RNAi, VC and NC groups (left) at day 7, respectively. (g) Quantification of pSmad1/5 protein levels in RNAi, VC and NC groups at day 3, 5 and 7, respectively. (h) Representative electrophoretic bands on the protein expression of CKIP-1, pSmad1/5, Smad1/5 and Smurf1 in RNAi, VC and NC groups at day 3, 5 and 7, respectively. (i) The mRNA expression levels of CKIP-1 and osteogenic marker genes (ALP, COL1, OPN, BSP and OCN) in Smad1 and Vector groups at day 3, 5 and 7, respectively. (j) The amount of deposited calcium in Smad1 and Vector groups (left) at day 7, respectively. (k) Quantification of pSmad1/5 protein levels in Smad1 and Vector groups at day 3, 5 and 7, respectively. (l) Representative electrophoretic bands on the protein expression of CKIP-1, pSmad1/5, Smad1/5 and Smurf1 in Smad1 and Vector groups at day 3, 5 and 7, respectively. Note: All data were mean ± s.d. *p < 0.05 for GT group vs. Control group, RNAi group vs. NC and VC group or Smad1 group vs. Vector group in the corresponding study.