Semi-quantitative analysis was carried out using ImageJ.1 Western blot analysis Cells were lysed in in 7?M urea, 0.1?M dithiothreitol (DTT), 0.05% Triton X-100, 25?mM NaCl and 20?mM 4-(2-hydroxyethyl)-1-piperazine ethane sulphonic acid (HEPES; pH 7.5). electrophoresis (SDS PAGE), transferred to Hybond-C nitrocellulose membrane (Amersham Pharmacia Biotech) and hybridised to an appropriate main antibody (Table 1), followed by horseradish peroxidase AC-55649 (HRP)-conjugated secondary antibodies: rabbit anti-mouse immunoglobulin G (IgG), rabbit anti-goat IgG and porcine anti-rabbit IgG (DakoCytomation; all at 1:1000 dilution). Immunoreactivity was detected by chemiluminescence. Table 1 Main antibodies utilized for immunolabelling. test and the MannCWhitney test AC-55649 were performed using Minitab. Data are expressed as means??standard deviation (SD). The criterion for significance was and compared to adherent cells and CD133? cells, respectively (Fig. 2G and H). Open in a separate windows Fig. 2 Characterisation of a subpopulation of CD133+ feline mammary carcinoma cells enriched for spheroid forming ability. A small population of CD133+ cells existing in feline mammary carcinoma cells were isolated by magnetic cell sorting. (A) CD133+ and CD133? cell fractions were processed and analysed for the expression of CD133 (120?kDa) by Western blot analysis. Single cells sorted for CD133 expression were evaluated for the potential to form spherical colonies in serum-free medium. Spheres created from CD133+ cells (B) but not CD133? cells (C). Level bars?=?20?M. (D) The numbers of the resultant spherical colonies from CD133+ and CD133? cells were counted. Data are representative of three impartial experiments (and -actin gene expression levels. CD247 (H) Quantification of RT-PCR results using ImageJ to determine the relative density of the bands compared to -actin loading controls. In vivo tumorigenic potential of mammospheres Single cells were expanded to 1 1??106 (at 2?h post-treatment (Fig. 6A). Open in a separate windows Fig. 6 Feline malignancy stem cells lack activation of the p53 DNA damage pathway in response to doxorubicin and ionising radiation. (A) Dissociated mammospheres and parental adherent cells were treated with 10?M doxorubicin AC-55649 or dimethyl sulfoxide (DMSO), cells were harvested over the indicated time course and expression of p53 pathway related proteins was assessed. (B) Dissociated mammospheres and parental adherent cells were treated with 5?Gy ionising radiation, cells were harvested over the indicated time course and expression of proteins related to the p53 pathway was assessed. Dissociated mammospheres and adherent cells were incubated with 2.5?M doxorubicin (Dox) or DMSO. Proteins were extracted according to their subcellular localisation: F1, cytosolic; F2, membranes/organelles; F3, nucleus; F4, nucleus; F5, cytoskeleton. Proteins from each portion were resolved by SDSCPAGE and analysed by immunoblotting for p53; 30?g was loaded per lane. Coomassie (CM) staining confirmed that protein expression profiles from each portion were unique; 5?g was loaded per lane (C). Levels of H2AX, an ATM target and a marker of DNA double strand breaks (Rogakou et al., 1998), similarly increased 2?h post-treatment in adherent cells (Fig. 6A), whereas in mammospheres treated with doxorubicin H2AX could not be detected and phosphorylation of p53-serine15 was delayed until 6?h post-treatment. Comparable results were obtained in response to irradiation (Fig. 6B). In untreated parental adherent cells, p53 protein was detected at a low level and was associated with the cytosolic and nuclear fractions, whereas in untreated mammospheres p53 protein levels were relatively high and were predominantly associated with membranes and, to a lesser extent, the nucleus (Fig. 6C). Upon DNA damage, the p53 protein levels in adherent cells increased in both the cytoplasmic and nuclear fractions, whereas in mammospheres p53 protein levels and subcellular localisation remain unchanged (Fig. 6C). Conversation The concept of CSCs is still evolving, but data implicating these cells in tumour maintenance and resistance to therapeutic brokers suggest that there is value in targetting CSCs in combination with standard therapies (Pang and Argyle, 2009). In this study, CSCs were isolated from a feline mammary carcinoma cell collection by mammosphere formation. The ability of mammospheres to grow in anchorage-independent conditions and to form three-dimensional spherical structures in serum-free.