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. 2017 Sep 19;7:11889. doi: 10.1038/s41598-017-12349-9

Figure 5.

Figure 5

Inhibitory effects of MI-2 on osteoclast formation through blockade of the NF-κB-NFATc1 signalling axis in monocytes. Inhibitory effects of MI-2 on A20 degradation and IKK α/β, p65, and IkBα phosphorylation in monocytes, as determined by in vitro osteoclast-differentiation assays. (a) Human monocytes were incubated in differentiation medium (α-MEM containing 10% FBS, 20 ng/mL MCSF, and 50 ng/mL RANKL) with or without MI-2. Protein expression in the cell lysates was analysed by immunoblotting with antibodies against pA20, phosphor-IKK α/β, and IKK α. β-actin expression was analysed as a loading control. The results shown are representative of 3 independent experiments. Full-length blots are presented in Supplementary Figure 4; (b) Human monocytes were incubated in differentiation medium (α-MEM containing 10% FBS, 20 ng/mL MCSF, and 50 ng/mL RANKL), with or without MI-2, at the indicated time points. Protein expression in the cell lysates was analysed by immunoblotting with antibodies against phosphor-p65 (p-p65), p65, phosphor-IkBα (p-IkBα), and IkBα. β-actin expression was analysed as a loading control. Full-length blots are presented in Supplementary Figure 4. The results shown are representative of 3 independent experiments. (c–e) The densities of p-p65, p65, p-IkBα, IkBα, p-Ikkα/β, and Ikkα/β bands were measured and normalised to β-actin levels. The p-p65/p65 and p-IkBα/ β-actin ratios are shown. Pooled results are shown from 3 independent experiments. * and ** indicate significant differences from control (untreated) cells based on 2-tailed unpaired Student’s t-tests at P < 0.05 and P < 0.01, respectively. The error bars show the s.e.m. values. (f) Human monocytes were incubated in differentiation medium with or without MI-2. NFATc1 protein expression in the cell lysates was analysed by immunoblotting with antibodies against NFATc1. β-actin expression was analysed as a loading control. Full-length blots are presented in Supplementary Figure 2. The results shown are representative of 3 independent experiments. (g–i) Total RNA was extracted from human monocytes incubated in differentiation medium with or without MI-2. NFATc1, MMP1, and MMP8 mRNA levels were measured by qPCR. Pooled results are shown from 3 independent experiments. ** and *** indicate significant differences based on 2-tailed unpaired Student’s t-tests at P < 0.01 and P < 0.001, respectively. The error bars show the s.e.m. values.