Evaluation of p-c-Jun (Ser63) levels and subcellular distribution in MAM-1 co-cultures treated with Iressa by A. Immunofluorescence and B. Flow Cytometry. MAM-1 were subcultured on coverslips or in 6-well plates and grown to ~95% confluence and then treated by replacing the conditioned media with fresh media that contained diluent (.001% DMSO, Control) or 1 μM Iressa for 1 hour prior to fixing and evaluation as described in the methods. A. Immunofluorescent photomicrographs were taken with the 100× objective under oil immersion of cells that were double labeled for p-c-Jun (Ser63) in red (TRITC) and HER2/neu in green (FITC) and counterstained with DAPI (blue) to define the nuclei. B. Flow cytometric analysis of control (left) and Iressa-treated (right) MAM-1 cultures. Cells were dual labeled for p-c-Jun (Ser63) and α-SMA. Density plots compare p-c-Jun (Ser63) expression levels in the α-SMA negative and positive subpopulations in control and Iressa-treated MAM-1 cultures. Mean Channel fluorescent values for p-c-Jun (Ser63) are indicated in the respective quadrants. Histogram analyses for p-c-Jun (Ser63) expression in the α-SMA negative subpopulations were generated from dot plots of p-Jun (Ser63)-PE versus Forward Scatter and gating on the respective the S (small) and L (large) fractions. Histograms of these tumor cell subpopulations demonstrate different baseline levels (Left) of p-c-Jun (Ser63) levels and Iressa responsiveness (Right) in small (S) and large (L) cells. Mean channel fluorescent values for p-c-Jun (Ser63) levels are indicated above the peaks. Parallel samples were dual stained for HER2/neu and p-c-Jun (Ser63) to verify these data (not shown).