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.