Detection of cells expressing stem cell markers or MUC1 in the lungs of MMT mice. (a) Immunocytochemical (ICC) staining. The colonies growing from lung tissue cells (as in Figure 1) were stained with anti-CD44 or Sca1 antibodies. The cells staining red were positive cells and those stained purple were considered double-positive cells (10x). The red square indicates the enlarged area. (b) Single-cell suspensions were made from the lungs of mice double transgenic for PyMT and MUC1 antigen (MMT) at the indicated ages and stained with monoclonal antibodies against CD44, Sca1 or MUC1 using ICC staining. Cells isolated from mammary tumors of MMT mice were used as positive control. Cells staining red were considered positive (60x). (c) Comparison of positive cells among different age groups (n = three to eight per group) of MMT and MT mice. The cells positive for the indicated antibody from each mouse were counted and are presented in the bar graph. Statistical significance between groups was determined using Chi-square test and the symbol "*" indicates P < 0.05 when 20 to 35 day group was compared with 53 to 87 day or 98 to 170 day groups. (d and e) Expression of CD24, ESA (epithelial specific antigen) or estrogen receptor (ER) on CD44/Sca1+ cells. The lung cells isolated from MMT mice at the indicated ages were processed for staining with anti-CD44-Cy, Sca1-FITC and CD24-PE, ESA-PE or ER-PE antibodies. The percentage of cells double positive for CD44 and Sca1 were gated and then further analyzed by FACS for expression of CD24, ESA or ER. The percentage of gated CD44/Scal+ cells in total lung or tumor cells and percentage of triple-positive cells are presented. (e) Mammary tumor cells isolated from 162-day-old MMT mice were used as controls.