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Figure 1 | BMC Cancer

Figure 1

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 1

The 4T1 CM inhibits MC3T3-E1 cell growth and differentiation, and enhances MC3T3-E1 cell apoptosis. (a) MC3T3 cells (2 × 104) were inoculated in 6-well culture dishes containing 10% FBS/AMEM and cultured for 12 h. After cell attachment, we changed the medium to 10% FBS/DMEM conditioned medium (CM) which had been pre-incubated with MC3T3-E1, 67NR, 66c14, 4T07, and 4T1 for 2 d, and kept culture for 5 days. Cells were harvested and counted under light microscopy every 2 days. n = 6, * p < 0.05, **p < 0.01, analyzed with t-test. (b) MC3T3-E1 cells (1 × 103) were inoculated in 96-well culture dishes and cultured in 10%FBS/AMEM medium for 12 h. After cell attachment, we changed the medium to MC3T3-E1, 67NR, 66c14, 4T07, and 4T1 CM, and kept culture for 7 d. Proliferation assays performed with WST-1 Assays. All groups compared with control group, n = 8, * p < 0.05, ** p < 0.01, analyzed with t-test. (c) The MC3T3-E1 cells were seeded at 8 × 104 cells/well in 6 well plates. Cells were maintained in MC3T3-E1, 67NR, 66c14, 4T07, and 4T1 CM for 21 days. The medium was changed every 3 d. After 21 d, cell lysates were processed to ALP ELISA Assay. All groups compared with control group, n = 4, * p<0.05, ** p<0.01, analyzed with t-test. (d) ALP ELISA Assay showed the ALP level of MC3T3-E1 cells cultured in 4T07 and 4T1 CM. Compared with 4T07 cells, n = 6, * p<0.05, ** p<0.01, analyzed with t-test. (e) MC3T3 cells (2 × 105) were inoculated in 6-well culture dishes containing 10% FBS/AMEM and cultured for 12 h. After cell attachment, we changed the serum free DMEM medium which had been pre-incubated with 4T07 and 4T1 for 2 d, and then cultured the MC3T3 cells for 3 d. Cells were harvested and counted under light microscopy every 2 days. Typical pictures showed that the medium pre-incubated with 4T1 cells enhanced MC3T3-E1 cell apoptosis. (f) After cultured in to serum free DMEM medium which had been pre-incubated with 4T07, and 4T1 for 2 d, the MC3T3 cells were kept culture for 1 d. Cells were analyzed with Annexin V and propidium iodide staining using flow cytometry. (g) Modified chemotactic Boyden chamber cell invasion assays (48 h) indicated that 4T1 cell line showed highest invasive ability among the 4 mouse breast cancer cell lines. Compared with 4T07 cell line, n = 4, * p<0.05, ** p<0.01, analyzed with t-test.

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