Fig 1: NMI levels in BALF A and ROC curve of NMI to predict severe CAP (B). Lower and upper lines indicate the 25th and 75th percentiles; middle lines indicate the 50th percentiles. *: P < 0.05, **: P < 0.01, ***: P < 0.001
Fig 2: Optimized effect of NMI on the PSI and CURB65 for predicting clinical outcomes. Comparison of ROC curves between N-PSI and PSI for predicting 30-day mortality A and ICU admission (B). ROC curves between N-CURB65 score and CURB65 score for 30-day mortality C and ICU admission (D)
Fig 3: Distribution of NMI levels by PSI class A and CURB65 score (B). Lower and upper lines indicate the 25th and 75th percentiles; middle lines indicate the 50th percentiles. **: P < 0.01, ***: P < 0.001
Fig 4: Kaplan–Meier survival curves by NMI cut-offs value and concentration stratification in CAP patients. Kaplan–Meier survival curves by NMI cut-offs value (55.48 pg/ml) for 30-day mortality A and ICU admission (B), and NMI concentration stratification (< 25 pg/ml, 25–50 pg/ml, 50–100 pg/ml, > 100 pg/ml) for 30-day mortality C and ICU admission (D)
Fig 5: NMI stimulates macrophages through the TLR4 pathway. a Western blot analysis of mNMI in the BMDM cell lysate after 1 h incubation with recombinant mNMI. The BMDM cells were isolated from Nmi -/- mice and pretreated with macrophage colony-stimulating factor (MCSF). b, c TNF and IL-6 released by BMDMs from WT (C57BL/6) mice, pretreated with bafilomycin A1 (10 nM), TAK-242 (100 nM) or dimethyl sulphoxide (DMSO) for 2 h and stimulated with mNMI (5 µg ml-1) or LPS (100 ng ml-1) for 8 h. d–g TNF and IL-6 levels in the supernatants of BMDMs from WT, Tlr4 -/- and Tlr2 -/- mice were analyzed using ELISA 4 h post activation by different stimulus. h After incubation with mNMI-GFP for 1 h, the percentage of GFP labeled CD11b+F4/80+ cells was determined by Flow cytometric analysis. The cells were isolated from spleen in WT or Tlr4 -/- mice. i NMI in the human THP1 cell (ATCC TIB-202™) lysates interacts with hTLR4. Ni-NTA beads coupled with 2 µg His-hTLR4 and/or His-hMD2 fusion proteins were used as bait. j The luciferase activity of HEK293T cells (ATCC CRL-11268™) are shown after stimulated with 5 µg ml-1 mNMI for 4 h (in the presence or absence of 25 µg ml-1 polymyxin B (PMB)). The cells were pre-transfected with mTLR4-MD2-CD14 and NF-?B promoter with luciferase activity. 100 ng ml-1 LPS was administrated as positive control. In b–e and g, error bars indicate ± s.e.m. from 3 biological replicates. Significance was tested by one-way ANOVA followed by Student–Newman–Keuls test. **P < 0.01
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