Figure 6

Versican G3 domain modulated MC3T3-E1 cell differentiation, growth and apoptosis via its epidermal growth factor-like motifs. (a) After culture the MC3T3-E1 cells in the bottom well of Transwell migration chambers for 12 h, vector, G3- and G3ΔEGF- transfected 4T07 cells (1 × 10⁵) were loaded in the insert pre-set with matrigel gel with 100 μl serum free DMEM medium and then incubated at 37°C for 48 hours. The invasive cells were stained blue and were counted in 6 fields of views/membrane using a light microscope. **, p<0.01. Error bars indicate SD (n = 6). (b) Vector-, G3- and G3ΔEGF- 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 1 ng/ml TGF-β1 for 5 d. Cells were counted by light microscope in 1, 3, 5 days. n = 6, * p<0.05, ** p<0.01, analyzed with t-test. (c) The G3- and G3ΔEGF-, and vector- transfected MC3T3-E1 cells were seeded at 8 × 10⁴ cells/well in a 6 well plate. Cells were maintained in 10% FBS/AMEM medium for 21 days. The medium was changed every 3 days. Cells were lysed 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) The G3- and G3ΔEGF- transfected MC3T3-E1 cells were maintained in 10% FBS/AMEM medium with 1 ng/ml TGF-β1 for 3 days. All cells were lysed and subjected to immunoblotting with antibodies to pEGFR, pSAPK/JNK, GSK-3β (S9P) and β-actin. (e) The G3- and G3ΔEGF-, and vector- transfected MC3T3-E1 cells (1 × 10³) were inoculated in 96-well culture dishes and cultured in 10%FBS/AMEM medium for 12 h. All samples were treated serum free AMEM medium with or without 2 ng/ml TNF-α for 7 days. The survival cells were counted under light microscope and compared with the seeded cells to present the viability (%). All groups compared with vector control cells, n = 9, * p<0.05, ** p<0.01, analyzed with t-test. The cell viability was assayed by WST-1 assays. (f) The G3- and G3ΔEGF- transfected MC3T3-E1 cells were treated with serum free AMEM medium with 2 ng/ml TNF-α for 24 hours. All samples were lysed and subjected to immunoblotting with antibodies to pEGFR, pSAPK/JNK, GSK-3β (S9P) and β-actin.