The Effect of the NFκB-USP9X-Cx43 Axis on the Dynamic Balance of Bone Formation/Degradation during Ossification of the Posterior Longitudinal Ligament of the Cervical Spine

Figure 5

ER stress involved in NF-κB/USP9X-mediated ossification of primary ligament fibroblasts. EPA (10 μM), 4-PBA (2 mM), or EPA plus PDTC (10 μM) was used to treat primary ligament fibroblasts, and Western blots showed that EPA treatment caused a significant USP9X increase, a Cx43 increase, a p65 decrease in the cytosol, and a p65 increase in nuclei. In contrast, 4-PBA treatment led to a significant USP9X decrease, a Cx43 decrease, a p65 increase in the cytosol, and a p65 decrease in nuclei (a). qPCR results showed that EPA treatment caused a significant USP9X increase while 4-PBA treatment caused a significant decrease of USP9X at the mRNA level. (c) EPA treatment caused a significant increase in ALP activity, while 4-PBA treatment caused a significant decrease in ALP activity. (d) Western blots showed that PDTC treatment reversed the EPA-induced USP9X increase, Cx43 increase, p65 decrease in the cytosol, and p65 increase in nuclei. (e) PDTC treatment reversed the EPA-induced USP9X increase at the mRNA level. (f) PDTC treatment reversed the EPA-induced increase of ALP activity. , vs. vehicle; ^## vs. EPA.

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