Figure 7. Transfection of siRNA into 4B12 cells.

(A) 4B12 cells were plated on 96-well plates at a cell density of 1 × 10³ per well, and cultured in α-MEM supplemented with 10% FBS and 30% CSCM. After 24 h, the cells were transfected with Cy3-labeled negative control #1 siRNA (20 nM) using TransIT-siQUEST transfection reagent (0.09 μl/well) according to the manufacturer’s protocol. TRAP(+) MNCs, which were formed from 4B12 cells in the presence of M-CSF (10 ng/ml) and sRANKL (10 ng/ml) for 7 days, were transfected with Cy3-labeled negative control #1 siRNA in the presence of M-CSF (10 ng/ml) and sRANKL (10 ng/ml). Transfection efficiency was estimated by counting the Cy3-positive cells at 48 h after transfection under a LSM 510 confocal microscope. The results are expressed as the mean ± standard deviation (SD) of quadruplicate cultures. (B) 4B12 cells were plated at a cell density of 5 × 10³ in each well of 96-well culture plates and cultured in α-MEM supplemented with 10% FBS and M-CSF (10 ng/ml) for 24 h, and transfected with siGFP (10 nM) and siTRAF6 (10 nM) using TransIT-siQUEST transfection reagent (0.09 μl/well). After 72 h, TRAF6, NFATc1 and TRAP expression were measured by qRT-PCR as described in Materials and Methods. Relative amounts of cDNA were calculated by the relative quantification (ΔΔCt) study method. The results are expressed as the mean ± SD of triplicate cultures. Similar results were obtained in two independent experiments. (C) After 5 days, TRAP activity was measured. Similar results were obtained in two independent experiments. (D) 4B12 cells were plated at a cell density of 5 × 10³ in each well of 24-well culture plates and cultured in the presence of M-CSF (10 ng/ml) plus sRANKL (10 ng/ml) plus hIL-1α (1000 u/ml) for 14 days, and transfected with siGFP (10 nM) and siTRAF6 (10 nM) using TransIT-siQUEST transfection reagent (0.09 μl/well). The trasfected cells were cultured in the presence of M-CSF plus sRANKL plus hIL-1α. After 72 h, the expression of TRAF6, NFATc1, and TRAP genes were measured. Similar results were obtained in two independent experiments.