Figure 5

RANKL plus M-CSF induced activation of ERK1/2 and Akt in C7 cells, which could be inhibited by minodronate and alendronate. (A, B) C7 cells were cultured in the presence of 25 ng/mL RANKL plus 50 ng/mL M-CSF for 15, 30, and 60 min (A) or 1, 3, and 10 days (B). (C, D) C7 cells were treated with 0.5 μM minodronate for 24 h. Cells were cultured in the presence of 25 ng/mL RANKL plus 50 ng/mL M-CSF for 15, 30, and 60 min (C) or 1, 3, and 10 days (D). (E, F) C7 cells were treated with 2 μM alendronate for 24 h. Cells were cultured in the presence of 25 ng/mL RANKL plus 50 ng/mL M-CSF for 15, 30, and 60 min (E) or 1, 3, and 10 days (F). Whole cell lysates were generated and immunoblotted with an antibody against phosphorylated ERK1/2 (phospho-ERK1/2), phosphorylated Akt (phospho-Akt), phosphorylated p38MAPK (phospho-p38MAPK), ERK1/2, Akt, and p38MAPK. (G–L) Quantification of the amount of phospho-ERK1/2, phospho-Akt, or phospho-p38MAPK normalized to the amount of total ERK1/2, Akt, or p38MAPK, respectively. The results are representative of 5 independent experiments. *P < 0.01 compared to controls. (M) ERK1/2, Akt, and p38MAPK activation in C7 cells, to which minodronate and alendronate were administered with or without the addition of GGOH. Phospho-ERK1/2, phospho-Akt, phospho-p38MAPK, ERK1/2, Akt, and p38MAPK levels were determined by immunoblotting analysis of the whole cell lysate. (N) Quantification of the amount of phospho-ERK1/2, phospho-Akt, or phospho-p38MAPK normalized to the amount of total ERK1/2, Akt, or p38MAPK, respectively. The results are representative of 4 independent experiments. *P < 0.01 compared to controls.