Regulatory Role of GSK-3β on NF-κB, Nitric Oxide, and TNF-α in Group A Streptococcal Infection
NZ131 infection induces the activation of NF-κB and the increased expression of iNOS and NO in macrophages. (a) Using immunocytochemical staining for detection of NF-κB p65 and DAPI for nuclear staining, the nuclear translocation of NF-κB in RAW 264.7 cells (2 × 105 cells/well in 24-well culture plate) after NZ131 infection (MOI: 10) at different time points was detected. Representative fields of NF-κB translocation in RAW 264.7 cells are shown. (b) The bar chart graph is the summary of (a) for the ratio of the change of NF-κB nuclear translocation after NZ131 infection at different time points. (c) RAW 264.7 cells were cotransfected with NF-κB promoter-driven luciferase reporter and Renilla luciferase-expressing plasmid for 24 h. Then, RAW 264.7 cells were infected with NZ131 (MOI: 10) for 1 h. Luciferase activity was used to determine the dynamic change of NF-κB activity 0.5–4 h after infection and the relative luciferase activity is the activity when compared with cells without NZ131 infection. (d) Western blot analysis was used to determine the expression of iNOS in RAW 264.7 cells stimulated by NZ131 (MOI: 10) for the indicated time points. (e) Griess reagent was used to determine the NO production in RAW 264.7 cells after treatment with different MOI of NZ131 (MOI: 0, 10, 50, or 100) at various time periods (6, 12, and 24 h after infection). (f) The expression of iNOS was determined by Western blot analysis with or without NF-κB inhibitor, PDTC (50 μM), for 6 h after NZ131 infection (MOI: 10). (g) The NO production was measured by Griess reagent 24 h after NZ131 infection (MOI: 10) with or without the concomitant treatment of PDTC. The data are means ± SD of the results obtained from three individual experiments. **; ***, comparisons between the indicated groups.