Journal: Nature Communications

Article Title: Denosumab attenuates knee osteoarthritis progression by inhibiting synovial inflammation via the RANK/TRAF6/FSTL1 signalling

doi: 10.1038/s41467-025-66202-z

Figure Lengend Snippet: A KEGG pathway analysis of DEGs, based on scRNA-seq. KEGG pathway enrichment was identified using a two-sided hypergeometric test. P -values were adjusted for multiple comparisons using the Benjamini-Hochberg false discovery rate (FDR) correction. Terms with an adjusted P -value (FDR) < 0.05 were considered statistically significant. B Western blot analysis of FSTL1, TRAF6, IκB, phosphorylated IκB, p65 and p50 proteins in FLSs. Cells were stimulated with RANKL or RANKL + denosumab for 24 h ( n = 3, biological replicates). C Western blot analysis of p65 and p50 proteins in the cytoplasm and nucleus of FLSs. Cells were stimulated with RANKL or RANKL + denosumab for 24 h. Cyto, cytoplasm; Nuc, nucleus ( n = 3, biological replicates). D Representative co-immunoprecipitation analyses. Protein lysates of FLSs were immunoprecipitated with anti-TRAF6 or non-specific (ns) IgG after stimulation with RANKL, or RANKL + denosumab. The pulldown was subjected to immunoblotting analysis against RANK of the coimmunoprecipitated protein and equal amounts of the total protein lysates (input). Representative images are from three independent experiments. E Western blot analysis of FSTL1, TRAF6, IκB, phosphorylated IκB, p65 and p50 proteins in the indicated FLSs ( n = 3, biological replicates). F Western blot analysis of p65 and p50 proteins in the cytoplasm and nucleus of indicated FLSs ( n = 3, biological replicates). G Schematic diagram of RANK/TRAF6/FSTL1-mediated NF-κB signalling regulated by RANKL. Created in BioRender. Hu, Y. (2025) https://BioRender.com/vva2i57 . H Representative immunofluorescence images of p65 (green) and p50 (red) in the indicated FLSs. Blue: DAPI showing total FLSs. Scale bar = 100 μm. Representative images are from three independent experiments. I Western blotting analysis of ADAMTS5, MMP-13 and TNF-α in the indicated FLSs ( n = 3, biological replicates). Source data and P values are provided in the Source Data file. Data are presented as median with interquartile range. Statistical analysis was performed using one-way ANOVA with Tukey’s post hoc test for multiple comparisons ( B , C , E , F ). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Cells cultured on slides were fixed with 4% PFA for 20 min at room temperature then incubated with a primary antibody against p50 (1:100, Proteintech, 14220-1-AP) or p65 (1:100, Proteintech, 10745-1-AP), CD86 (1:100, Proteintech, 13395-1-AP) or Arg1 (1:100, Affinity Biosciences, DF6657), overnight at 4 °C.

Techniques: Western Blot, Immunoprecipitation, Immunofluorescence

Journal: Redox Biology

Article Title: 5-Methoxytryptophan attenuates hypobaric hypoxia induced acute lung injury by alleviating lipid peroxidation via targeting peroxiredoxin 6

doi: 10.1016/j.redox.2025.103922

Figure Lengend Snippet: Hif1α promoted the nuclear translocation of NF-κB p50 to inhibit Asmt. A-B. qPCR detected the relative mRNA level of Asmt in PMVECs; C .The immunoblotting and quantitative data showed the effect of si-Hif1α on Asmt in PMVECs; D. ELISA measured the effect of si-Hif1α on the level of 5-MTP in the supernatants of PMVECs. E. The motif and possible binding sites of NF-κB p50 in the promoter region of Asmt; F. ChIP-qPCR detected the binding of NF-κB p50 in Asmt; G . Immunofluorescence staining and quantitative data of NF-κB p50 nuclear translocation. Scale bar, 25 μm; H . qPCR detected the effect of PDTC (30 μM, 30min) on the relative mRNA level of Asmt in PMVECs; I. The immunoblotting and quantitative data showed the effect of PDTC (30 μM, 30min) on Asmt in PMVECs; J. ELISA measured the effect of PDTC (30 μM, 30min) on the level of 5-MTP in the supernatants of PMVECs. A-J. Values were mean ± SEM for n = 3; A. ∗ p < 0.05, compared with the Control group; B-D. ∗ p < 0.05, compared with the Normoxia + si-NC group; # p < 0.05, ## p < 0.01, compared with the Hypoxia + si-NC group; F. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, compared with the corresponding IgG group. ### p < 0.001, compared with the corresponding Control group; G-J. ∗∗ p < 0.01, compared with the Control + PBS group; # p < 0.05, ## p < 0.01, compared with the Hypoxia + PBS group. qPCR, quantitative polymerase chain reaction; PMVECs, mouse pulmonary microvascular endothelial cells; ChIP, chromatin immunoprecipitation; ELISA, enzyme-linked immunosorbent assay; NC, negative control; PDTC, pyrrolidinedithiocarbamate. Statistical significance was evaluated using t -test for A and one-way ANOVA for B-J.

Article Snippet: Primary antibodies against NF-κB p50 (Proteintech, 14220-1-AP, 1:500), Prdx6 (Proteintech, 13585-1-AP, 1:1000) and Lamp2 (Abcam, ab13524, 1:500) were diluted in Immunol Staining Primary Antibody Dilution Buffer (Beyotime) and incubated overnight at 4 o C. PBS was used for negative control.

Techniques: Translocation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Binding Assay, ChIP-qPCR, Immunofluorescence, Staining, Control, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Negative Control

Journal: Redox Biology

Article Title: 5-Methoxytryptophan attenuates hypobaric hypoxia induced acute lung injury by alleviating lipid peroxidation via targeting peroxiredoxin 6

doi: 10.1016/j.redox.2025.103922

Figure Lengend Snippet: Hif1α promoted the nuclear translocation of NF-κB p50 to inhibit Asmt. A-B. qPCR detected the relative mRNA level of Asmt in PMVECs; C .The immunoblotting and quantitative data showed the effect of si-Hif1α on Asmt in PMVECs; D. ELISA measured the effect of si-Hif1α on the level of 5-MTP in the supernatants of PMVECs. E. The motif and possible binding sites of NF-κB p50 in the promoter region of Asmt; F. ChIP-qPCR detected the binding of NF-κB p50 in Asmt; G . Immunofluorescence staining and quantitative data of NF-κB p50 nuclear translocation. Scale bar, 25 μm; H . qPCR detected the effect of PDTC (30 μM, 30min) on the relative mRNA level of Asmt in PMVECs; I. The immunoblotting and quantitative data showed the effect of PDTC (30 μM, 30min) on Asmt in PMVECs; J. ELISA measured the effect of PDTC (30 μM, 30min) on the level of 5-MTP in the supernatants of PMVECs. A-J. Values were mean ± SEM for n = 3; A. ∗ p < 0.05, compared with the Control group; B-D. ∗ p < 0.05, compared with the Normoxia + si-NC group; # p < 0.05, ## p < 0.01, compared with the Hypoxia + si-NC group; F. ∗∗ p < 0.01, ∗∗∗ p < 0.001, ∗∗∗∗ p < 0.0001, compared with the corresponding IgG group. ### p < 0.001, compared with the corresponding Control group; G-J. ∗∗ p < 0.01, compared with the Control + PBS group; # p < 0.05, ## p < 0.01, compared with the Hypoxia + PBS group. qPCR, quantitative polymerase chain reaction; PMVECs, mouse pulmonary microvascular endothelial cells; ChIP, chromatin immunoprecipitation; ELISA, enzyme-linked immunosorbent assay; NC, negative control; PDTC, pyrrolidinedithiocarbamate. Statistical significance was evaluated using t -test for A and one-way ANOVA for B-J.

Article Snippet: The remaining chromatin was immunoprecipitated overnight at 4 °C with 5 μg primary antibody against Hif1α (GeneTex, GTX127309), NF-κB p50 (Proteintech, 14220-1-AP), Histone H3 (CST, #4620) or rabbit IgG (CST, #2729).

Techniques: Translocation Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Binding Assay, ChIP-qPCR, Immunofluorescence, Staining, Control, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Negative Control