Fig 2: Importance of timing of kinase inhibition during HAstV1 infection. (A)Time course of activation of MAPKs and Akt. Lysate was prepared from either mock-infected (Mock) or HAstV1-infected (Infected) Caco-2 cells at designated time points. The lysates were subjected to Western blot to detect either total kinases (ERK, p38, JNK, and Akt) or their phosphorylated forms [(pERK, pp38, pJNK, and pAkt) MAPKs and Akt]. The two bands in the “ERK” and “pERK” panels represent ERK1 (44 kDa) and ERK2 (42 kDa), respectively. The two bands in “JNK” and “pJNK” panels represent JNK1 (54 kDa) and JNK2 (46 kDa), respectively. (B)Effect of inhibitors on kinase activation. Lysates prepared at designated time points from HAstV1-infected Caco-2 cells in the presence of solvent alone (DMSO), LY294002, or U0126 were subjected to Western blot for either total ERK (ERK) or its phosphorylated form (pERK). ERK1 and 2 bands are indicated by an arrowhead and an arrow, respectively. (C)Quantitative presentation of the Western blot signal shown in A. Digital images of the bands were quantitated using ImageJ. The vertical line indicates the relative value of the signal intensity divided by the value of the band for each mock-infected sample, at 0.25 hpi, in each experimental group. The black and gray bars represent values for the mock- and HAstV1-infected sample, respectively. Note that, due to large differences, the scale of the vertical bar representing “Akt” differs from that of others. (D)Effects of inhibitors on capsid protein expression. Caco-2 cells were infected and examined by immunofluorescence analysis as in Figure 1, except that solvent alone or inhibitors were added at indicated time points and maintained until fixing at 24 hpi. For each time point, the proportions of cells positive for capsid protein expression were examined statistically as described in Methods. (*P < 0.05; **P < 0.001).

Fig 3: Curcumin up-regulated BK protein expression via ERK1/2 signaling pathway.(A) Effects of curcumin on ERK1/2, JNK, p38 phosphorylation. HEK293 cells were incubated with 10µM curcumin for 24 h, and the phosphorylation of ERK1/2, JNK, p38 were analyzed by Western Blot. (B) Graphic representation of densitometric data of the phosphorylation level of ERK1/2 (n = 3). (C) Western Blot analysis showed that the ERK1/2 inhibitors, U0126 (10 µM), abolished the effect of curcumin on BK protein. Inhibitors were applied 1 h before administration of curcumin. (D) Graphic representation of densitometric data show a protein level of BK (n = 4). * p < 0.05; ** p < 0.01; n.s., not significant.

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: 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