Effect of Iressa on (A) PCNA activity and (B) Signal transduction in the tumor and stromal cell populations of MAM-1 co-cultures. A. MAM-1 co-cultures were treated for 24 h with fresh media in the absence (Control) or presence of 1 μM Iressa then fixed and stained with PC10-TRITC to detect PCNA reactivity (red) and counterstained with DAPI to identify the nuclei (blue) Photomicrographs were taken with the 100× objective under oil immersion. The PCNA index of control tumor cells is >95% and <12% in the Iressa treated tumor cells. In the stroma adjacent to the Iressa treated tumor nest, PCNA index is >95%. Note the presence of apoptotic cells in DAPI stained tumor cells treated with Iressa (arrow). B. Differential effect of Iressa on PCNA, phospho-p44/42 MAPK (Thr202/Tyr204) and phospho-MEK1/2 (Ser217/221) in the ErbB-2 Negative (Stromal) and ErbB-2 Positive (Tumor) populations in MAM-1 treated for 24 h with 1 μM Iressa. Samples were dual-labeled for ErbB-2 and the indicated antigen and evaluated by dual-color flow cytometry. Bars represent the Mean Channel Fluorescent Values for phospho-p44/42 MAPK (Blue Bars), phospho-MEK1/2 (Red Bars) or PCNA (Black Bars). Following Iressa treatment, the ErbB-2 positive population decreased by 44% and the α-SMA positive population increased 3-fold in these MAM-1 co-cultures.