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. 2015 Dec 1;5:17605. doi: 10.1038/srep17605

Figure 6. XN suppresses RANKL-induced NF-κB signaling pathway, but has little effect on MAPK/AP-1 signaling.

Figure 6

(A) The effect of XN on RANKL-induced IκBα degradation and p65 phorsphorylation. RAW264.7 cells were pretreated with XN (5 μM) for 3 hours, and then stimulated with RANKL (30 ng/ml) for indicated time. The degradation of IκBα and the phosphorylation of p65 were tested by Western blot analysis (left). The Western blot were performed in triplicate. The IκBα protein level (with β-actin for normalization) were quantified by Quantity One software (right). (B) The effect of XN on RANKL-induced p65 nuclear translocation. RAW264.7 cells were pretreated with different doses of XN for 3 hours, and then stimulated with RANKL for 20 minutes. Cell Nuclear Extracts (NE) and Cytoplasmic Extract (CE) were collected and subjected to Western blot analysis with the indicated antibodies. (C) The effect of XN on RANKL-induced activity of NF-κB. RAW264.7 cells were co-transfected with NF-κB-luciferase reporter gene and Renilla gene. After 48 hours, the cells were treated with RANKL and indicated concentrations of XN for another 24 hours. Cell extracts were collected and luciferase activity was measured. (D) NF-κB (p65) prevents the inhibitory effect of XN in RANKL-induced osteoclast differentiation. RAW264.7 cells were transfected with p65 or control plasmids, and then incubated with or without XN (5 μM) in the presence of RANKL (30 ng/ml). After 4 days, the cells were stained for TRAP activity and the numbers of osteoclasts were counted. (E) XN has little effect on RANKL-induced phosphorylation of MAPKs. BMMs were cultured in the presence of XN (5 μM) for 4 hours, and then RANKL was stimulated at the indicated time points. Cell lysates were extracted for Western blot analysis with indicated antibodies. (F) XN has little effect on the activity of AP-1 induced by RANKL. RAW264.7 cells were co-transfected with AP-1-luciferase reporter gene and Renilla gene. After 48 hours, the cells were treated with RANKL and indicated concentrations of XN for another 24 hours. Cell extracts were collected and luciferase activity was measured. Column, means of experiments conducted in triplicate; bar, SD. *p < 0.05, **p < 0.01, ***p < 0.001.