Fig 2: Mirt2 inhibits TRAF6 oligomerization and auto-ubiquitination. a Silver-stained SDS-PAGE gel analysis of proteins in macrophages that are bound to biotinylated lncRNA-Mirt2. The highlighted regions were analyzed by mass spectrometry, identifying TRAF6 as a protein unique to Mirt2. b Immunoblotting analysis of proteins in macrophages bound to biotinylated Mirt2 using anti-TRAF6 antibody. c RNA immunoprecipitation (RIP) analysis to determine the recovery of Mirt2 in macrophages using anti-TRAF6 antibody. IgG served as control. Data represent the mean ± SEM of three independent experiments. *P < 0.05 vs. IgG group. d RNA FISH technology and immunofluorescent analysis to determine the co-localization of Mirt2 (green) and TRAF6 (red) in macrophages. DAPI was used for nucleus staining (blue). The images correspond to a single slice of Z-stacks and the experiments were repeated three times. e RNA pull-down analysis was employed to determine the interaction of TRAF6 with full-length or truncations of Mirt2. f Constructs for myc-tagged TRAF6 (wild type or domain truncation mutants) were transfected into HEK293T cells, pulled down by biotinylated Mirt2 transcript and examined by Western blot with antibody to myc. Bottom: the domain structure of TRAF6. g Immunoprecipitation of HA-TRAF6 (myc-TRAF6) followed by immunoblot analysis of myc-TRAF6 (HA-TRAF6) in HEK293T cells expressing both TRAF6 constructs to detect TRAF6 oligomerization in the presence or absence of Mirt2. h Immunoblot analysis of the ubiquitination of TRAF6 in HEK293T cells transfected to express myc-tagged TRAF6 and HA-tagged ubiquitin. Left panel: Total ubiquitination of TRAF6; Middle panel: K48-linked and K63-linked ubiquitination of TRAF6; Right panel: The effects of different concentrations of Mirt2 on the ubiquitination of TRAF6. i Immunoblot analysis of the K63-linked ubiquitination of endogenous TRAF6 in macrophages. j Immunoprecipitation of HA-Ubc13 (myc-TRAF6) followed by immunoblot analysis of myc-TRAF6 (HA-Ubc13) in HEK293T cells expressing both constructs to detect the interaction of TRAF6 and Ubc13. k Immunoblot analysis of the content of Ubc13 immunoprecipitated using TRAF6 antibody in macrophages. l Effects of Mirt2 knockdown on the phosphorylation of p65 and Jnk in macrophages transfected with si-TRAF6 or scramble siRNA, as determined by Western blot

Fig 3: Mirt2 attenuates LPS-induced endotoxemia. Endotoxemia was induced in C57BL/6 mice by intraperitoneal injection of LPS (25 mg/kg), and control animals were administered with equivalent volumes of normal saline. Adenovirus (Ad-Mirt2 or Ad-EV) were delivered into mice by tail veil injection 3 days before LPS challenge. a Adenovirus infection efficiency at 72 h after adenovirus administration. b Survival curve of mice with endotoxemia (n = 12). c Levels of cytokines (IL-1-ß, IL-6 and TNF) in serum of mice challenged with LPS for 6 h. Data are expressed as mean ± SEM (n = 8). *P < 0.05 vs. Ad-EV group. d Histopathology in lung of Ad-EV or Ad-Mirt2 treated mice 24 h after LPS challenge. Upper panel: Hematoxylin and eosin staining; Middle panel: Immunohistochemical staining for macrophage marker F4/80; Lower panel: Immunohistochemical staining for neutrophil marker Ly6G. e Quantitative analysis of lung injury in panel d. Left panel: Lung injury score; Middle panel: Quantitative analysis of F4/80 positive cells; Right panel: Quantitative analysis of Ly6G positive cells. Data are expressed as mean ± SEM (n = 8). *P < 0.05 vs. Ad-EV group. f Western blot analysis for the phosphorylation of p65 and Jnk in lung of endotoxemia mice. g Quantification of band density in panel f. Data are expressed as mean ± SEM (n = 5). *P < 0.05 vs. Ad-EV group. h Histopathology in liver of Ad-EV or Ad-Mirt2 treated mice 24 h after LPS challenge. Upper panel: Hematoxylin and eosin staining; Middle panel: Immunohistochemical staining for macrophage marker F4/80; Lower panel: Oil Red O staining. i Quantitative analysis of liver injury in panel h. Left panel: Liver injury score; Middle panel: Quantitative analysis of F4/80 positive cells; Right panel: Determination of the contents of triglyceride. Data are expressed as mean ± SEM (n = 8). *P < 0.05 vs. Ad-EV group. j Western blot analysis for the phosphorylation of p65 and Jnk in liver of endotoxemia mice. k Quantification of band density in panel j. Data are expressed as mean ± SEM (n = 5). *P < 0.05 vs. Ad-EV group. Two-tailed Student’s t-test for two groups

Fig 4: MAPKs are the downstream pathway of TLR2 critical for phagocytosis and phagosome maturation of NPCs. (A,B) Western blot analysis of MAPKs (ERK, JNK, p-38) in NPCs from WT and Tlr2-/- mice infected with S. aureus for different time periods. * P < 0.05, the group of WT vs. the group of Tlr2-/-, **P < 0.05, the different time point in WT group or Tlr2-/- group. (C) FACS analysis to detect the interaction between NPCs and S. aureus with or without MAPKs specific inhibitors pretreatment. *P < 0.05, the S. aureus group vs. the different inhibitors group. (D) Co-localization of S. aureus and Lyso-Tracker Red was detected by fluorescence micrograph. P values were determined by T-test and one-way ANOVA. All data are presented as the means ± S.D from three independent experiments.

Fig 5: Analysis of regulation of MAPKs in HepG2 cells treated with non-cytotoxic AgNPs and Ag+.(A) Cells were treated with a series of concentrations of 10 nm AgNPs, 100 nm AgNPs or Ag+ for 24 hours, and the samples were analyzed with dual-phospho-ERK (Thr202/Tyr204) antibody, ERK antibody, dual-phospho-p38 (Thr180/Tyr182) antibody, p38 antibody, dual-phospho-JNK (Thr183/Tyr185) antibody and JNK antibody, respectively, using western blot. ß-actin was used for equal loading. The panel shows images representative of three independent experiments. (B, C, D) The intensity of each chemiluminescence protein band was analyzed by ImageJ and normalized to the MAPKpp/ß-actin ratio (control = 1), with the data expressed as mean ± S.D. (n = 3), *p<0.05.