Fig 2: Antagonistic effect of si-NLRP3 on the inhibition of the osteogenesis of hPDLSCs by TNF-α. (A) Protein level of NLRP3 in hPDLSCs transfected with si-NLRP3 or si-NC was examined by western blot analysis. **P<0.01 vs. si-NC. (B) Protein levels of p-p65, p65, p-IκBα and IκBα were measured by western blot analysis. (C) Band intensities were quantified using ImageJ software. **P<0.01, ***P<0.001 vs. control + si-NC; ##P<0.01 vs. TNF-α + si-NC, NS (no significant difference) vs. TNF-α + si-NC. (D) Protein levels of COL1, ALP and RUNX2 on day 7 of osteogenic induction. (E) Band intensities were quantified using ImageJ software. **P<0.01 vs. control + si-NC; ##P<0.01 vs. TNF-α + si-NC. (F) mRNA expression levels of COL1, ALP and RUNX2 following osteogenic induction for 7 days. **P<0.01 vs. control + si-NC; #P<0.05, ##P<0.01 vs. TNF-α + si-NC. (G) ALP staining following osteogenic induction for 7 days (scale bar, 100 μm). (H) ALP activity analysis on day 7 of osteogenic induction. **P<0.01 vs. control + si-NC; ##P<0.01 vs. TNF-α+si-NC.

Fig 3: SARS-CoV-2 N protein promotes NF-κB signaling at TAK1 and IKK complex level. a Schematic overview of NF-κB signaling pathway. b HEK293T cells were transiently transfected with NF-κB luciferase reporter, Flag-empty vector (EV) and increasing amount of Flag-N protein together with plasmids expressing IRAK1, TRIF, RIPK1, TRAF2, TRAF6, TAK1 and TAB1, IKKβ and p65. Cell lysates were collected at 36 h post-transfection and luciferase activities were tested. c HEK293T cells were transiently transfected with HA-N protein plasmid along with Flag-empty vector (EV), Flag-TAK1, Flag-TAB1, and Flag-TAB2 plasmids, followed with 1 μg/mL poly(I:C) treatment for 6 h before harvested. Whole-cell lysates were subjected to immunoprecipitation with anti-Flag beads and immunoblot analysis was performed with indicated antibodies. d HEK293T cells were transiently transfected with HA-N protein plasmid along with Flag-EV, Flag-IKKα, Flag-IKKβ, and Flag-NEMO plasmids, followed with 1 μg/mL poly(I:C) treatment for 6 h before harvested. Whole-cell lysates were subjected to immunoprecipitation with anti-Flag beads and immunoblot analysis was performed with indicated antibodies. e Extracts of Calu3 cells infected with SARS-CoV-2 at MOI = 0.1 for 48 h were subjected to immunoprecipitation with anti-SARS-CoV-2 N protein and immunoblot analysis with indicated antibodies. f–g Representative images (f) of confocal microscopy of co-localization between SARS-CoV-2 N protein and IKKβ in Huh7 cells with SARS-CoV-2 infection at MOI = 0.2 for indicated time points followed by labeling of N protein, IKKβ, and DAPI. Scale bars, 5 μm. Quantitative analysis (g) of the puncta between IKK and SARS-CoV-2 N protein (10 cells per sample). Data in (b) are expressed as mean ± SEM of at least three independent experiments. Data in (g) are expressed as mean ± SD. *p < 0.05, **p < 0.01, ns not significant (two-tailed Student’s t-test). For (c–f), similar results are obtained for three independent biological experiments

Fig 4: Sal attenuated TNF-α-driven inflammation in human chondrocytes via inhibition of NF-κB signaling. (A–C) The levels of NO, PGE2, and IL-6 in the culture medium of human OA chondrocytes pretreated with Sal at different concentrations and stimulated with 20 ng/ml TNF-α. (D, E) The levels of iNOS and COX-2 in human chondrocytes pretreated with Sal at different concentrations and stimulated with 20 ng/ml TNF-α. (F, G) Cytoplasmic IκBα and nuclear p65 levels in human chondrocytes pretreated with Sal at different concentrations and stimulated with 20 ng/ml TNF-α. (H) The nuclear translocation of p65 was detected using immunofluorescence combined with DAPI staining of nuclei (scale bar: 20 µm). All results are presented as the means ± SDs of six duplicate experiments. ##P < 0.01 compared to the control group; *P < 0.05, **P < 0.01 compared to the TNF-α alone group.

Fig 5: Effect of Quer on the activation of the NF-κB/NLRP3 pathway in TNF-α-induced hPDLSCs. hPDLSCs were treated with 1 μM Quer, 20 ng/ml TNF-α, or their combination for 24 h in a CM. (A) Protein expression levels of p-p65, p65, p-IκBα and IκBα. (B) Band intensities were quantified using ImageJ software. **P<0.01 vs. control; #P<0.05, ##P<0.01 vs. TNF-α. (C) Protein expression levels of NLRP3, procaspase-1 and caspase-1. (D) Band intensities were quantified using the ImageJ software. **P<0.01 vs. control; #P<0.05, ##P<0.01 vs. TNF-α. (E) mRNA expression levels of NLRP3 and caspase-1. **P<0.01 vs. control; #P<0.05, ##P<0.01 vs. TNF-α. hPDLSCs, human periodontal ligament stem cells; Quer, quercetin; TNF-α, tumor necrosis factor-α; NLRP3, NOD-like receptor family pyrin domain-containing protein 3.