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. 2017 Oct 3;7:12600. doi: 10.1038/s41598-017-12707-7

Figure 2.

Figure 2

NUBML act as a negative regulator of osteoclastogenesis via inhibiting NF-κB activation. Bone marrow cells were isolated from WT mice and transfected with pMX-GFP and pMX-NUMBL. Two day after transfection, BMMs cells were plated in different plates and treated with RANKL and M-CSF for osteoclastogenesis, actin ring staining, mRNA isolation and western blot. (A,B) TRAP staining and (C) actin ring formation assay shows that NUMBL expression inhibits OC differentiation. (D) Increased NUMBL expression results in decreased expression of OC differentiation associated genes (F) TRAP, (F) NFATc1, (G) DC-STAMP, and (H) MMP9, (I) Cathepsin-K and (J) β3-Integrin in bone marrow cells upon RANK stimulation when compared with GFP expressing cells. (K) Western blots suggest decreased NFATc1 expression in NUMBL overexpressing cells in response to RANKL stimulation when compared with GFP expressing control cells. β-Actin was used as control for both qPCR and Western blotting. Full-length Western blots images are included in supplementary data as Figure S2.K. (L) Bone marrow cells isolated from RelA-Luc reporter mice were transfected with pMX-GFP and pMX-NUMBL and cultured in presence of M-CSF for 3 days, followed by RANKL stimulation for 0, 15, 30 and 60 minutes. RelA-luciferase activity measurement shows that NUMBL overexpression results in decrease in NF-κB activation in comparison to GFP expressing cells upon RANKL stimulation. (M) NUMBL expression was confirmed by western blotting for Flag. (**P < 0.01 and ***P < 0.001).