Figure 4

Effects of MK CM on osteoblastic proliferation and differentiation. (a) Viability of pre-osteoblast MC3T3-E1 cells was assessed using a CCK-8 assay in the presence or absence of 50% (v/v) conditioned media (CM) from megakaryocytes (MK) or pro-megakaryocytes (pro-MK) for 48 hours. MK and pro-MK cells were derived from K562 cells. (b,c) Viability of MC3T3-E1 cells was also determined with the indicated doses of phorbol 12-myristate 13-acetate (PMA) (b) or 50% (v/v) CMs of enriched MK and pro-MK derived from murine fetal livers (c) for 48 hours. (d) Proliferation of MC3T3-E1 cells was assessed using a BrdU incorporation assay in the presence or absence of 50% (v/v) MK and pro-MK CMs for 48 hours. (e,f) Alkaline phosphatase (ALP) activity (e) and osteocalcin secretion (f) of MC3T3-E1 cells in the presence or absence of 30% (v/v) MK and pro-MK CMs for 7 days. The ALP activity was normalized by total cellular protein amounts. (g) Bone nodule formation assay of MC3T3-E1 cells was assessed by Alizarin red S staining, and were quantified by extraction with cetylpyridinium chloride in the presence or absence of 30% (v/v) MK and pro-MK CMs for 14 days. MK and pro-MK were derived from K562 cells, unless otherwise specified. Data are presented as mean ± SEM. *P < 0.05 vs. non-CM (α-MEM media); ^#P < 0.05 vs. pro-MK CM. NS, not significant.