Figure 4
MG132 suppresses HSV-1-mediated NF-κB activation. (a) Effects of HSV-1 infection and MG132 treatment on NF-κB signaling. Vero cells were infected with HSV-1 at an MOI of 1 and cultured in media in the presence or absence MG132 for 0.4, 12, 24, or 36 hours. Whole-cell lysates were subjected to immunoblotting with an anti-IκBα or -NF-κB (p65) antibody. The values of IκBα/β-Actin are presented at the bottom of the image, and the values of 0.4 h nontreated and noninfected cells are presented as 1.0. (b) Immunofluorescence analysis using anti-IκBα antibody. Vero cells were mock infected (i) or HSV-1 infected at an MOI of 1 (ii, iii, iv) for 30 min and were treated with DMSO (i, ii), 0.025 μM MG132 (iii) or 0.75 μM MG132 (iv) for 12 hours. Cells were fixed with 4% PFA for 20 min and permeabilized with 0.1% Triton X-100 in PBS for 10 min, followed by incubation with anti-IκBα (green) antibody and DAPI to stain the nuclei (blue). Cells were observed by a fluorescence microscope (IX71) with a 60x oil-immersion objective. Scale bars represent 20 μm. The single channel gray images of IκBα are shown in Supplementary Fig. S3. (c,d) MG132 inhibits HSV-induced NF-κB transcriptional activity. HeLa cells were transfected with the NF-κB (p65) reporter plasmid and cultured for 18 hours. Transfected cells were infected with HSV-1 at an MOI of 1 for 30 min and treated with various concentrations of MG132 for 12 hours (c) or 24 hours (d), followed by cell lysis for luciferase detection. The NF-κB activity of HSV uninfected and MG132-untreated cells was defined as 100 relative activity. Standard deviations were determined by three independent experiments and are indicated by the error bars. (e) Subcellular localization of p65 and IκBα in HSV-1 infected cells. Vero cells were infected with HSV-1 at an MOI of 1 and then cultured with 1.0 or 1.5 μM MG132 for 7 hours. Harvested cells were lysed, and cytoplasmic fraction (C) and nuclear fraction (N) were prepared. The p-PTEN and lamin B1 were used as the cytoplasmic marker and the nuclear marker, respectively. Original images of blotting data (a and e) are shown in Supplementary Fig. S3.
