Multinucleation in p53-/- Clara cells a) Primary cultured Clara cells immunohistochemically labelled with anti-CC10 (blue). Expression of the Clara cell specific marker CC10 (Clara cell 10 kDa protein) was observed in cultures derived from both wild type and p53-/- lungs. In p53-/- cultures, two or more nuclei were frequently observed in a single CC10-positive cell (arrows). b) Primary cultured Clara cells immunohistochemically labelled with anti-BrdU. Clara cells were cultured in the presence of BrdU from plating to 24 hours culture, from 24 to 48 hours culture, and from 48 to 72 hours culture. Nuclei that had incorporated BrdU during S phase DNA synthesis were detected with an anti-BrdU antibody (brown). In p53-/- cultures, two or more nuclei that had incorporated BrdU were frequently observed in a single cell (arrows). c) Both wild type and p53-/- Clara cells displayed similar rates of S phase DNA synthesis over the first 3 days in culture, as determined by counts of BrdU incorporation (BrdU index = no. of BrdU positive nuclei / total no. of nuclei, expressed as a percentage). d) The number of multinucleated cells in p53-/- cultures increased as a feature of time in culture (multinucleation index = no. of multinulceated cells / total no. of cells, expressed as a percentage). The data represents mean ± SD (n = 3), * indicates p < 0.05 (t test).