Rstained with TOPRO). (B) DDR checkpoint activation. Similar sections were evaluated

Rstained with TOPRO). (B) DDR checkpoint activation. Similar sections were evaluated for their activation of p53, by assaying the nuclear accumulation of pS15-p53. Basement membranes are demarcated by the dotted line, and examples of nuclei positive for pS15-p53 are shown (B, basal; L, luminal). (C) pS15-pGenotoxins Inhibit Wnt-Dependent Mammary Stem Cellactivation in response to DMBA. To evaluate the activation of DDR in response to DMBA, compared to other more canonical genotoxins, lysates of mammary epithelial cells (HC11 cells) treated with various genotoxins, were evaluated by Western blotting. The genotoxins were: 10 Gy irradiation (1 hour), 2 mg/ml DMBA (24 hours; this is a pro-genotoxin requiring activation by metabolism, or vehicle, DMSO), N-ethyl-N-nitrosourea (1 hour after addition of 500 mg/ml ENU, a direct 11967625 acting alkylating agent, or control PCB), or 20 mg/ml camptothecin (1 hour, camptothecin is a topoisomerase I inhibitor, or DMSO). Similarly, the specificity of the nuclear stain was cross-checked to Western blot data of lysates of primary mammary epithelial cells in culture, exposed to DMBA (as indicated). Non-treated cells (NT) are also shown for comparison. (D) Induction of proliferation is equally allocated to basal and luminal cells after exposure to DMBA. Genotoxin-exposed and control-treated mice (1, 2 and 7 weeks after treatment) were administered BrdU (intraperitoneally), and mammary glands harvested 2 hours later. Data that describes the lineage-specific mitotic index is shown for samples 2 weeks after DMBA treatment. Thus, the fraction of BrdU-positive basal and luminal cells was assayed (n = 3, .2000 cells each), and illustrated here as a fraction of each cell type (top panel) or as a fraction of total cells (basal cells are a minority). Statistically Danusertib site different values are indicated * (p,0.05). (E) Induction of proliferation is focalized after exposure to DMBA. Genotoxin-exposed and control-treated mice (1, 2 and 7 weeks after treatment) were administered BrdU (intraperitoneally), and mammary glands harvested 2 hours later. Mitotic cells were widely distributed in vehicle-treated glands. Though this pattern was also evident in DMBA-administered glands, in addition, there were “bursts” of mitotic activity, defined as five or more BrdU positive cells in a 20-cell radius (B). Bursts were measured per length of duct (n = 3; arbitrary unit of cm after image capture). doi:10.1371/journal.pone.0049902.gDMBA exposure. In the presence of Wnt Dipraglurant site ligand, the overall rate of growth was not significantly different (Fig. 6C). However, although Wnt did not change the relative ability of luminal and basal cells to activate H2AX throughout the activation phase, these two cell types showed vastly different responses during the cellular response phase (48 hours after treatment). Indeed, there were no remaining basal cells with nuclear H2AX (Fig. 6C). This suggests that only Wnt-responsive basal cells (and not basal cells in the presence of EGF/2 serum) showed a selective cell death response to the DDR. The activation of canonical Wnt signaling was assessed byassay of Axin2 mRNA (Fig. 6D), and this was not suppressed by DDR/DMBA administration. As a marker of p53 activation, p21 mRNA expression was analyzed, and showed significant induction in cultured cells 24 hours after exposure to DMBA. During the response phase, cells expressing p21 were eliminated. Basal cells were observed to accumulate in the presence of Wnt3a, using linea.Rstained with TOPRO). (B) DDR checkpoint activation. Similar sections were evaluated for their activation of p53, by assaying the nuclear accumulation of pS15-p53. Basement membranes are demarcated by the dotted line, and examples of nuclei positive for pS15-p53 are shown (B, basal; L, luminal). (C) pS15-pGenotoxins Inhibit Wnt-Dependent Mammary Stem Cellactivation in response to DMBA. To evaluate the activation of DDR in response to DMBA, compared to other more canonical genotoxins, lysates of mammary epithelial cells (HC11 cells) treated with various genotoxins, were evaluated by Western blotting. The genotoxins were: 10 Gy irradiation (1 hour), 2 mg/ml DMBA (24 hours; this is a pro-genotoxin requiring activation by metabolism, or vehicle, DMSO), N-ethyl-N-nitrosourea (1 hour after addition of 500 mg/ml ENU, a direct 11967625 acting alkylating agent, or control PCB), or 20 mg/ml camptothecin (1 hour, camptothecin is a topoisomerase I inhibitor, or DMSO). Similarly, the specificity of the nuclear stain was cross-checked to Western blot data of lysates of primary mammary epithelial cells in culture, exposed to DMBA (as indicated). Non-treated cells (NT) are also shown for comparison. (D) Induction of proliferation is equally allocated to basal and luminal cells after exposure to DMBA. Genotoxin-exposed and control-treated mice (1, 2 and 7 weeks after treatment) were administered BrdU (intraperitoneally), and mammary glands harvested 2 hours later. Data that describes the lineage-specific mitotic index is shown for samples 2 weeks after DMBA treatment. Thus, the fraction of BrdU-positive basal and luminal cells was assayed (n = 3, .2000 cells each), and illustrated here as a fraction of each cell type (top panel) or as a fraction of total cells (basal cells are a minority). Statistically different values are indicated * (p,0.05). (E) Induction of proliferation is focalized after exposure to DMBA. Genotoxin-exposed and control-treated mice (1, 2 and 7 weeks after treatment) were administered BrdU (intraperitoneally), and mammary glands harvested 2 hours later. Mitotic cells were widely distributed in vehicle-treated glands. Though this pattern was also evident in DMBA-administered glands, in addition, there were “bursts” of mitotic activity, defined as five or more BrdU positive cells in a 20-cell radius (B). Bursts were measured per length of duct (n = 3; arbitrary unit of cm after image capture). doi:10.1371/journal.pone.0049902.gDMBA exposure. In the presence of Wnt ligand, the overall rate of growth was not significantly different (Fig. 6C). However, although Wnt did not change the relative ability of luminal and basal cells to activate H2AX throughout the activation phase, these two cell types showed vastly different responses during the cellular response phase (48 hours after treatment). Indeed, there were no remaining basal cells with nuclear H2AX (Fig. 6C). This suggests that only Wnt-responsive basal cells (and not basal cells in the presence of EGF/2 serum) showed a selective cell death response to the DDR. The activation of canonical Wnt signaling was assessed byassay of Axin2 mRNA (Fig. 6D), and this was not suppressed by DDR/DMBA administration. As a marker of p53 activation, p21 mRNA expression was analyzed, and showed significant induction in cultured cells 24 hours after exposure to DMBA. During the response phase, cells expressing p21 were eliminated. Basal cells were observed to accumulate in the presence of Wnt3a, using linea.