Figure 2. Aberrantly proliferating cells in RB null lungs progress into S-phase and mitosis.

Immunohistochemical analysis for BrdU incorporation (A) and PH3 (B) in RB ablated and control adult lungs from mice treated with doxycycline throughout gestation. Statistically significant increases in BrdU and PH3 positive cells (arrows) were noted in RB ablated versus control lungs on day 14 after injury (*p=0.001 and *p=0.005, respectively). A minimal increase in percent BrdU positive cells was seen on day 0 in RB ablated versus control lungs (*p=0.02), but no significant increase was seen in percent PH3 positive cells on day 0 (p=0.39). Quantitative analysis is presented as average + SEM. Data is representative of 6–7 animals per time point. Double label immunohistochemical analysis for BrdU (brown nuclear stain, open arrowhead) and CCSP (blue cytoplasmic stain, closed arrowhead) in RB ablated and control adult lungs from mice treated with doxycycline throughout gestation (C). The majority of BrdU incorporating cells were CCSP positive (arrow) in the RB ablated and control lungs on day 4 after injury (BrdU-CCSP double positive/BrdU positive cells = 80.1 ± 16.1 and 71 ± 14.9, respectively). The increase in S-phase Clara cells was maintained in RB ablated lungs on day 14 after injury (BrdU-CCSP double positive/BrdU positive = 81.4 ± 6.6). The overall labeling index (BrdU positive/total epithelial cells) in control lungs at day 14 after injury was 1.6 ± 1.5 which is comparable to baseline levels before injury. Quantitative data represents average ± SD. Percent positive cells was calculated counting >250 BrdU positive epithelial cells representing 3–5 animals per time point. br=bronchiole; Original magnification: 1000×