Figure 4

WFA pre-treatment inhibits nuclear translocation of NFκB subunit p65 in KKLEB cells stimulated with PAF. Cells were plated out, treated and immunostained for fluorescence microscopy in optical-bottom 96-well plates as described in "Findings". Images of fixed cells were captured using a Discovery 1 system at 20× magnification under identical conditions across all sample wells. The images were not digitally processed prior to analyses. Six images were analyzed per group. Representative images are shown. For presentation, images were imported into Image J (NIH, Bethesda, MD) and pseudocolored. A FITC-conjugated antibody was used to detect p65 (left set of columns, pseudocolered green) and DAPI was used to delineate the nuclei of each cell (middle set of columns, pseudocolored red). Merged images are shown in the right column. Images are grouped as follows (top to bottom rows): Control cells treated with media containing 10% LB Broth (LB), cells treated with 10% PAF (PAF), cells pre-treated with WFA (3 μM) followed by stimulation with 10% PAF (WFA + PAF). Translocation of p65 to the nucleus is clearly noted upon treatment with PAF. Translocation of p65 is inhibited by WFA pre-treatment leading to decreased FITC-associated fluorescence in nuclei of KKLEB cells as demonstrated in the bottom row.