Figure 1. NOD2 was up-regulated in DENV2-infected and L18-MDP-transfected THP-1 cells. (A) Confocal micrograph of THP-1 macrophage-like cells having undergone the following treatments for 12 h: infection with DENV2, transfection with L18-MDP, or untreated (mock control). The cells were prepared by immunostaining for NOD2 (green ) and nuclear contrast (blue ). The micrograph (on the right) illustrates the entire field of each treatment, and a cell positive for NOD2 is depicted in the box. Images in the upper panels denote the percentage of NOD2 positive cells for each condition (N=110). The pictures (magnification 2100x) were cropped to improve the presentation. The bar graphs (B and C) portray the NOD2 expression in cells at distinct time points post-infection under different treatments: Mock (white bars), transfected with L18-MDP (gray bars), or infected with DENV2 at an MOI of 10 (black bars). The bar graphs indicate the degree of increase (fold change) calculated by the ratio of NOD2/GAPDH (D) and NOD2/β-actin (E). (B) mRNA of NOD2 was measured at 3, 6 and 12 h post-infection, using end point RT-PCR with specific primers for human NOD2 and GAPDH. (C) Relative NOD2 protein expression was established by Western blot analysis of the whole-cell lysates with an anti-NOD2 antibody. An anti-β-actin antibody served as the loading control. Representative gel and membrane images from RT-PCR and western blot at different time points are shown. Significance was determined with one-way ANOVA: *** p<0.001, ** p<0.05, * p<0.05, and ns: not significant.
Expression of RIP2 and MAVS effectors and co-localization with NOD2
RIP2 was constitutively expressed in all experimental groups, including the untreated cells (Fig. 2A). Its expression was more abundant than that of NOD2 (Fig. 2D). At 6 hpi, DENV2-infected cells and L18-MDP-transfected cells both displayed an enhanced RIP2 expression (Figs. 2B and C). At this time point, co-localization of NOD2 and RIP2 was found in ~25% of the DENV2-infected cells (Fig. 2H), while co-localization was limited or absent in the mock-infected or transfected cells (Figs. 2G and I). At 12 hpi, the expression of the MAVS effector was absent in untreated cells (Fig. 3A), marginal in DENV2-infected cells (Fig. 3B), and slightly higher (than the infected group) in PIC-transinfected cells. At the same time point, the increased expression of MAVS in the transfected cells correlated with the expression of NOD2 in the cytosol (Figs. 3C, F and I), and some co-localization between MAVS and NOD2 was observed in the DENV2-infected cells (Fig. 3H). A quantitative measure of cells showing some overlapping was carried out to have a more objective value using different microscopy fields as indicated in Suppl. Fig. 2.