Fig 1: Serum expression of S100A8 (a), S100A9 (b), TfR1 (c), and SAP (d) detected by ELISA in the different clinical groups, respectively. Increased S100A8 and S100A9 serum levels were observed for all PQ poisoning patients (n = 21) compared to healthy controls (n = 21). Conversely, PQ poisoning groups showed significantly lower serum levels of TfR1 and SAP than the healthy group.
Fig 2: IHC analysis of S100A8 and S100A9 expression in lung tissue from PQ-treated rat and healthy controls. (a) and (b) Absence of staining for S100A8 and S100A9 in the normal lung tissue, respectively. (c) and (d) Marked S100A8 and S100A9 staining in lung tissue from rat treated with PQ. (Original magnification, 40×. Scale bars = 50 µm). Results of S100A8 (e) and S100A9 (f) immunohistochemistry in the cytoplasm and cell membrane in the lung tissues were quantitated by average optical of positive staining per 200 field.
Fig 3: Receiver-operating characteristic (ROC) analysis for S100A8 (a), S100A9 (b), TfR1 (c), and SAP (d) to discriminate PQ poisoning patients from healthy controls, respectively.
Fig 4: Increased Level of S100A8 Secreted by RNF5-Deficient IECs Affects Host Immune Responses(A) Immunoblot analysis of endogenous proteins in MODE-K cells expressing empty vector (EV) or two RNF5-targeting shRNAs.(B) Representative images of IHC staining for S100A8 and S100A9 (left) and quantification of IHC scores (right) in colon sections from WT and Rnf5-/- mice (n = 6, two fields each). Scale bars, 50 µm. ****p < 0.0001 by two-tailed t test.(C) Immunoblot analysis of MODE-K cells expressing EV, shRNF5, or shRNF5 plus shS100A8.(D) ELISA quantification of S100A8 levels in the culture supernatants of MODE-K cells expressing EV, shRNF5, or shRNF5 plus shS100A8 (n = 3).(E) ELISA quantification of IL-12p70 and IL-1ß levels in the culture supernatants of bone marrow-derived dendritic cells (BMDCs) fromWT mice incubated for 18 hr with medium alone (no stimulation), recombinant S100A8 (1 ng/mL), or conditioned medium (CM) from MODE-K cells expressing EV, shRNF5, or shRNF5 plus shS100A8 (n = 3).(F) MFI of MHC classes I and II on CD11c+ BMDCs incubated as in (E) (n = 3).(G) Relative luciferase activity of MODE-K-EV, MODE-K-shS100A8, MODE-K-shRNF5, or MODE-K-shRNF5/shS100A8 cells transiently transfected with the NF-?B-dependent firefly luciferase reporter plasmid NF-?B-luc and Renilla luciferase plasmid pRL-TK (n = 3).All data are representative of three independent experiments. Graphs show mean ± SEM. n.s., not significant (p > 0.05). *p < 0.01, **p < 0.001, and ***p < 0.0001 by one-way ANOVA followed by Tukey’s multiple comparison test (D–G).
Fig 5: S100A8 Is a Substrate of RNF5(A) Immunoprecipitation and immunoblot analysis of the RNF5-S100A8 interaction in HEK293T cells ectopically expressing Flag-tagged RNF5 or empty vector (EV) and V5-tagged S100A8.(B) Immunoprecipitation and immunoblot analysis of HEK293T cells co-expressing V5-S100A8, HA-ubiquitin (HA-Ub), plus Flag-RNF5. Cells were treated withMG132 (10 µM) for 4 hr prior to lysis.(C) Anti-S100A8 immunoprecipitation and anti-Ub immunoblot of MODE-K cells transfected with EV or RNF5-targeting shRNA and treated with MG132 (10 µM) for 4 hr before lysis.(D) Immunoblot analysis of HEK293T cells expressing Flag-EV or Flag-RNF5 and treated with medium or 10 µM MG132 for 4 hr before lysis.(E) Immunoblot analysis of MODE-K cells expressing EV or shRNF5. Cells were treated with cycloheximide (CHX) as indicated (n = 3).(F) Immunoprecipitation and immunoblot analysis of the interaction between endogenous RNF5 with S100A8 analyzed in MODE-K cells treated with MG132 (10 µM) for 4 hr.(G) Immunoprecipitation and immunoblot analysis of cell lysates prepared from MODE-K cells treated with 10 ng/mL TNF-a for the indicated times followed by MG132 (10 µM) for 4 hr before lysis.Data are representative of three independent experiments. Graphs show mean ± SEM.
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