Figure 3

Expression of versican G3 domain regulated MC3T3-E1 cells growth and differentiation. (a) Vector- and G3- transfected MC3T3 cells (2 × 10⁴) were inoculated in 6-well culture dishes containing 10% FBS/AMEM and cultured for 12 h. After cell attachment, the cells were cultured with or without 1 ng/ml TGF-β1 for 5 d. Cells were also counted by light microscope in 1, 3, 5 days. n = 6, * p<0.05, ** p<0.01, analyzed with t-test. (b) The G3- and vector-transfected MC3T3-E1 cells (1 × 10³) were inoculated in 96-well culture dishes and cultured in AMDM medium containing 1 ng/ml TGF-β1 or not for 7 days. Proliferation assays performed with WST-1 assay. All groups compared with vector control cells, n = 8, * p<0.05, ** p<0.01, analyzed with t-test. (c) The vector- and G3-transfected MC3T3-E1 cells were seeded at 8 x10⁴ cells/well in a 6 well plate. Cells were maintained in 10% FBS/AMEM medium containing 1 ng/ml TGF-β1 for 21 days. The medium was changed every 3 days. Cells were lysised and processed to ALP ELISA Assay. All groups compared with vector control cells, n = 6, * p<0.05, ** p<0.01, analyzed with t-test. (d) Typical pictures showed ALP staining of vector- and G3-transfected MC3T3-E1 cells maintained in 10% FBS/AMEM medium containing 1 ng/ml TGF-β1 for 21 days. (e) The vector- and G3-transfected MC3T3-E1 cells were maintained in 10% FBS/AMEM medium with or without 1 ng/ml TGF-β1 for 3 days. All cells were lysed and subjected to immunoblotting with antibodies to pEGFR, pAKT, pSAPK/JNK, GSK-3β (S9P) and β-actin.