Fig 2: circNFKB1 regulates NFKB1 expression and sustains NF-?B signaling pathway.a qRT-PCR analysis showing NFKB1 mRNA levels after circNFKB1 knockdown in HCs. b Western blotting analysis showing p105, p50 and p65 phosphorylation protein levels in circNFKB1 knockdown chondrocytes. c qRT-PCR analysis showing NFKB1 mRNA levels after circNFKB1 overexpression in HCs. d Western blotting analysis showing p105, p50, and p65 phosphorylation protein levels in circNFKB1 overexpressed chondrocytes. e Immunofluorescence staining of NFKB1 (upper panels) and p-p65 (lower panels) of chondrocytes in the indicated groups at 8 weeks after first injection or DMM surgery (n = 5 mice). Scale bars, 50 µm. f Quantification of NFKB1 and p-p65 positive cells were performed according to staining results (n = 5 per group). a–d, f, mean values ± SD are shown, and p values were calculated by two-tailed Student’s t test (c, d, f) or one-way ANOVA followed by Sidak’s multiple comparison test (a, b). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. b, d, e, representative images are shown.

Fig 3: Schematic illustration of how HBsAg interferes with the nuclear factor kappa B (NF-?B) signaling pathway. HBsAg specifically binds to TAK1 and TAB2, suppresses the polyubiquitination of TAK1-TAB2 complex, blocks TAK1 and IKBa phosphorylation, inhibits the NF- ?B subunits p50 and p65 translocation from cytoplasm to nucleus, and downregulates the expression of IFNs and inflammatory cytokines. On the other hand, HBsAg interferes the molecular interaction between TAK1 and TAB2 with promoting autophagic degradation, further inhibiting the activation of NF-?B signaling pathways.

Fig 4: Vpr-mediated regulation of miR-210-5p expression.(A) Promoter analysis using a series of reporter plasmids involving the miR-210-5p promoter (intact, f1; truncated, f2; mutated, f3) with pCMV-Flag-Vpr in HOS cells. (B) p50 and p-p50 protein levels were determined by western blot analysis in MT4 cells inoculated with HIV-1 or HIV-1 Vpr- virus. The relative p-p50 or p50 levels were estimated by densitometry; the ratios of p-p50 were calculated relative to p50. (C) Nucleus-p50 (N-p50) or cytoplasm-p50 (C-p50) protein levels were determined by western blot analysis in MT4 cells inoculated with HIV-1 or HIV-1 Vpr- virus. The relative N-p50 or C-p50 levels were estimated by densitometry; the ratios of N-p50 were calculated relative to C-p50. (D) HOS cells were transfected with vector or Vpr for 30 h and were assessed by ChIP assay, as described in the Materials and Methods. The data are presented as mean ± SD for three biological replicates, and statistical significance compared to the controls was calculated by t test. **P < 0.01, ***P < 0.001, NS: not significant.

Fig 5: miR-9-5p suppresses inflammatory factors and the NF-?B signaling pathway in 16HBE cells. (A) Q-PCR analysis of the IL-6, IL-8, TNF-a and lncRNA RP11-86H7.1 mRNA levels after miRNA (miR-9-5p, miR-17-5p, miR-31-5p, and miR-221-3p) mimic transfection. (B) Q-PCR analysis of NFKB1 levels after miR-9-5p mimic transfection and miR-9-5p mRNA levels after knockdown of the lncRNA RP11-86H7.1. (C) ELISA analysis of IL-6, IL-8, and TNF-a levels in 16HBE cells after miR-9-5p mimic transfection. (D) Western blotting analysis of the protein levels of I??ß, p-I?B-a, I?B-a, p-p65, p65 and NFKB1 after miR-9-5p mimic transfection. GAPDH was used as an internal control. The data are shown as the means ± standard deviations (n = 3). The statistical significance of the data was assessed by Student’s t test. *P < 0.05, **P < 0.01.