Figure 5

Expression of versican G3 domain regulated MC3T3-E1 cells apoptosis via EGFR/JNK pathway. (a) G3- and vector-transfected MC3T3-E1 cells (2 × 10⁵) were inoculated in 6 well culture dishes with 10% FBS/AMEM medium. After cultured for 12 hours, all samples were treated serum free AMEM medium with or without 2 ng/ml TNF-α for 4 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 = 6, * p<0.05, ** p<0.01, analyzed with t-test. (b) G3- and vector-transfected MC3T3-E1 cells (1 x 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. 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. (c) G3- and vector-transfected MC3T3-E1 cells were treated with serum free AMEM medium with 2 ng/ml TNF-α for 24 hours and processed to Annexin V assays. (d) G3- and vector-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. (e) G3- and vector-transfected MC3T3-E1 cells were treated with serum free AMEM medium with 2 ng/ml TNF-α, with or without 100 nM SP60025 for 4 days. Cell viability was assessed by WST-1 assays. The cell viability was presented by absorbance value of treated samples compared with absorbance value of same groups 12 hours after loading (%). The survived cells were counted under light microscope and compared with the seeded cells to present the viability (%). (f) G3- and vector-transfected MC3T3-E1 cells were treated with serum free AMEM medium with 2 ng/ml TNF-a, with or without 100 nM SP60025 for 24 hours. All samples were lysed and subjected to immunoblotting with antibodies to pSAPK/JNK, GSK-3β (S9P) and β-actin.