Fig 2: SC inhibits the NF-κB signalling pathway by promoting TRAF2 expression. A, B The interaction between TRAF2 and P65 and between TRAF2 and IκBα was predicted by the HDOCK server online. C, D Marc-145 cells were seeded in 12-well plates and transfected with siTRAF2 (50 nM), siNC (negative control), pXJ40-HA-TRAF2, or vector. Cells were harvested at 36 hpi after transfection to detect the expression levels of TRAF2, P65, p-P65, IκBα, p-IκBα, P100 and P52. E Marc-145 cells were seeded in 12-well plates and cultured for 12 h. The cells were infected with PRRSV SD-YL1712 (MOI = 1) and collected at 24 and 36 hpi for Western blotting to determine the expression levels of TRAF2 and p-TRAF2. F Marc-145 cells in 12-well plates were infected with PRRSV SD-YL1712 (MOI = 1) and then treated with 10 µM SC. Cells were harvested at 36 hpi for Western blotting to determine the expression levels of TRAF2, p-TRAF2, and PRRSV N.

Fig 3: SMF promoted IL-6 secretion from PDLSCs under mechanical stress. (A) RT-PCR assay of expression levels of IL-1β, TNF-α, IL-6, RANKL, OPG, and RANKL/OPG relative to GAPDH in PDLSCs after 24 h. Data for each gene was processed using the 2ΔΔCt method. (B) ELISA of IL-6 protein in PDLSCs supernatant after 24 h. (C) RT-PCR was performed to evaluate the efficiency of IL-6 inhibition (*P < 0.05). (D,E) Representative TRAP images showing osteoclast formation after seven days of induction. (F,G) Immunohistochemistry of IL-6 expression levels on the pressure side of tooth movement. AOD integrated optical density/area; n = 3; data are mean ± SD; *P < 0.05 vs Con; #P < 0.05 vs OTM + sham.

Fig 4: SMF promoted osteoclast formation by PDLSCs under mechanical stress. (A) Schematic diagram of the in-vitro models. (B) Morphology of PDLSCs stimulated by mechanical loading and SMF after 24 h. (C) Schematic diagram of osteoclast formation induced by the PDLSCs supernatant. (D,E) Representative TRAP images showing osteoclast formation after 7 days of induction. (F,G) Western blot image and semi-quantitative measurement of TRAP expression in osteoclasts after 7 days of induction. n = 3; ns, not significant; **P < 0.01 vs Con group; #P < 0.05 vs Mech. loading group.

Fig 5: PRRSV Nsp10 interacts with TRAF2 and decreases its expression. A CRL-2843 cells were transfected with pEGFP-C-PRRSV nonstructural proteins for 36 h and then harvested to determine the expression of pEGFP-C-Nsps and TRAF2. B HEK-293T cells were cotransfected with p3×Flag-Nsp10 and pXJ40-HA-TRAF2. After 36 h, the cells were collected, and Co-IP was used to determine the interaction between the Nsp10 protein and TRAF2. C HEK-293T cells were transfected with pXJ40-HA-TRAF2 and p3×Flag-Nsp10. After 36 h, the cells were collected to determine the reverse Co-IP interaction between TRAF2 and Nsp10. D HEK-293T cells were cotransfected with the p3×Flag-Nsp10 and pXJ40-HA-TRAF2 or p3×Flag-Nsp11 and pXJ40-HA-TRAF2 proteins. After 24 h, the cells were stained with anti-Flag and anti-HA antibodies to determine the position of Nsp10 (green) relative to TRAF2 (red), and images were captured by confocal microscopy.