Figure 6

U0126 (MEK1/2 inhibitor) or LY294002 (PI3K inhibitor) inhibited osteoclast formation. (A, B) C7 cells were treated with 0.25, 0.5, 1, or 2.5 μM U0126 (A) or 0.5, 1, 2.5, or 5 μM LY294002 (B). Cells receiving U0126 or LY294002 were cultured in the presence of 25 ng/mL RANKL plus 50 ng/mL M-CSF. Cultures were fed every 3 days by replacing with 500 μL of fresh medium, with or without U0126, LY294002, RANKL, and M-CSF. Cultures were fixed and stained for TRAP-positive multinucleated cells per well was counted. These results are representative of 5 independent experiments. *P < 0.01 compared to 25 ng/mL RANKL plus 50 ng/mL M-CSF administration. (C, D) C7 cells were treated with 0.5, 1, or 2.5 μM U0126 or 1 or 5 μM LY294002 (D) 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) 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. (E, F) Quantification of the amount of phospho-ERK1/2 or phospho-Akt normalized to the amount of total ERK1/2 or Akt, respectively. The results are representative of 5 independent experiments. *P < 0.01 compared to controls. (G, H) C7 cells were treated with 0.5, 1, or 2.5 μM U0126 or 1 or 5 μM LY294002 (D) for 24 h. Cells were cultured in the presence of 25 ng/mL RANKL plus 50 ng/mL M-CSF for 1, 3, and 10 days. (I, J) Quantification of the amount of phospho-ERK1/2 or phospho-Akt normalized to the amount of total ERK1/2 or Akt, respectively. The results are representative of 5 independent experiments. *P < 0.01 compared to controls.