MEK5 siRNA inhibited the formation of TRAP (+) MNCs in 4B12 cells.

<p>(A) 4B12 cells were transfected with GFP siRNA or MEK5 siRNA. The 4B12 cells (2.5 × 10⁴) were cultured in the presence of M-CSF (10 ng/ml) and sRANKL (20 ng/ml). After 3 days, MEK5 gene expression was measured by qRT-PCR. Similar results were obtained in two independent experiments. *<i>P</i><0.05 when compared with 4B12 cells transfected with GFP siRNA. (B) 4B12 cells (5 × 10⁵) were transfected with siRNAs for GFP or MEK5. The levels of MEK1, 2, and 5 were analyzed by Western blot analysis. Similar results were obtained in two independent experiments. (C) 4B12 cells were transfected with siRNAs for GFP or MEK5. The cells were stimulated with M-CSF (10 ng/ml). After 30 min, the phosphorylation of ERK5 was monitored. Similar results were obtained in two independent experiments. (D) 4B12 cells (5 × 10⁵) were transfected with GFP or MEK5 siRNAs. The transfected cells (5 × 10³) were cultured in the presence of M-CSF (10 ng/ml) and sRANKL (20 ng/ml). After 6 days, the number of TRAP-positive MNCs was counted. Similar results were obtained in three independent experiments. *<i>P</i><0.05 when compared with 4B12 cells transfected with GFP siRNA.