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. Author manuscript; available in PMC: 2017 Jun 15.
Published in final edited form as: J Immunol. 2016 May 9;196(12):4977–4986. doi: 10.4049/jimmunol.1502044

Figure 2. Inhibition of miR-182 significantly suppresses TNF-α-induced osteoclastogenesis in RBP-J-deficient cells.

Figure 2

BMMs derived from RbpjΔM/ΔM mice were transfected with either control or miR-182 antagomir (40 nM) and were stimulated with TNF-α for 3 days. TRAP staining was performed (A) and the number of TRAP-positive MNCs (>= 3 nuclei per cell) per well relative to the control was calculated (B). Data in figure represent average ± SEM of three independent experiments and statistical significance was calculated by paired-t-test. **P<0.01. TRAP-positive cells appear red in the photographs. Bar, 200 µm. (C) Quantitative real-time PCR analysis of mRNA expression of Acp5 (encoding TRAP), Ctsk (encoding cathepsin K) and Itgb3 (encoding β3) in BMMs from RbpjΔM/ΔM mice transfected with control or miR-182 antagomir (40 nM) and stimulated with or without TNF-α for 3 days. Data are representative of and statistical analysis was performed on at least three independent experiments. *P<0.05. **P<0.01. (D) Immunoblot analysis of NFATc1 and Blimp1 expression in whole cell lysates obtained from RbpjΔM/ΔM BMMs transfected with either control or miR182 antagomir (40 nM) in the absence or presence of TNF-α for 3 days. p38α was measured as a loading control. (E) The relative density of the immunoblot bands of NFATc1 and Blimp1 vs. those of loading control p38α from three independent experiments were quantified by densitometry and normalized to the unstimulated control condition. *P<0.05. (F) Cell proliferation/viability at 0, d1, d2 and d3 with control or miR-182 antagomir (40 nM) transfection was examined by MTT assay (refer to Methods for details). (G) Toluidine blue-stained dentin resorption pits (left panel) formed by the osteoclasts derived from RbpjΔM/ΔM BMMs transfected with control or miR-182 antagomir (40 nM) in the presence of TNF-α (40 ng/ml) for 10 days. Bar, 200 µm. Relative pit area (% to slice) is shown in the right panel. Data are representative of and statistical analysis was performed on three independent experiments. *P<0.05.