Figure 6

rMtb9.8 increases the nuclear and total amounts of phosphorylated NF-κB p65. RAW264.7 cells were cultured in the presence of rMtb9.8 (5 µg/ml) for the indicated times (0, 10, 30 min), after which a total of 1 × 10⁴ cells were stained as described in the Materials and Methods section; images of the cells were acquired using the ImageStream 100. The nuclear fluorescence intensity and total fluorescence intensity of phosphorylated p65 were analyzed using IDEAS 6.0 software. The cells were stained for phosphorylated p65 and with the nuclear dye DAPI following rMtb9.8 activation; the histograms show the nuclear fluorescence intensity (A) and the total fluorescence intensity of phosphorylated p65 (B) of cells incubated with rMtb9.8 for the indicated times. (C) The nuclear fluorescence intensity and total fluorescence intensity of phosphorylated p65 following rMtb9.8 activation for 0, 10 and 30 min were analyzed by fluorescence intensity (FI) using IDEAS 6.0 software. The results show that the nuclear fluorescence intensity of phosphorylated p65 and the total fluorescence intensity of phosphorylated p65 increased significantly at 30 min after rMtb9.8 induction compared with that in unstimulated cells. (D) The nuclear-to-cytoplasmic ratio (N/C ratio) of phosphorylated p65 following rMtb9.8 activation was analyzed by fluorescence intensity (FI) using IDEAS 6.0 software. The results show that the mean and SEM of the N/C ratio of phosphorylated p65 dramatically increased by 30 min following rMtb9.8 activation as measured by fluorescence intensity (FI). The data shown represent one of three independent experiments. **P < 0.01, ***P < 0.001, stimulated cells versus cells cultured in medium alone.