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Figure 6 | BMC Cancer

Figure 6

From: The role of versican G3 domain in regulating breast cancer cell motility including effects on osteoblast cell growth and differentiation in vitro– evaluation towards understanding breast cancer cell bone metastasis

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.

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