Fig. 5

MMTV-Spy1 mice show alterations in DDR pathway when exposed to DMBA. a Schematic of short-term DMBA treatment and collection of samples. b Western blot for Spy1 (left panel) and p53 (right panel) levels in 8-week-old control mice and DMBA-treated mice 48 h following DMBA exposure. Densitometry analysis is depicted with total Spy1 and p53 levels corrected for total levels of Actin. c Immunohistochemical analysis for Nedd4 expression in inguinal mammary glands of 8-week-old MMTV-Spy1 mice and littermate controls was performed after exposure to DMBA. Representative images are shown in the left panel. Levels of Nedd4 were quantified using ImageJ analysis (right panel). Scale bar = 100 μm. d Representative images of immunohistochemical analysis of γH2AX in inguinal mammary glands of 8-week-old MMTV-Spy1 and littermate control (Cntl) mice after exposure to DMBA (left panel), where brown stain is γH2AX and blue stain is haematoxylin. Number of γH2AX-positive cells were counted and quantified as percentage of γH2AX cells (right panel). Scale bars = 100 μm and 50 μm (inset image). e Primary mammary epithelial cells from MMTV-Spy1 mice and control littermates were isolated and UV irradiated with 50 J/m². Cells were collected 0, 1, 3, 6 and 24 h post UV, and immunofluorescence was performed to assess formation of γH2AX foci following damage (n = 3). f HC11 cells were transfected with pCS3 and Myc-Spy1-pCS3, and UV irradiated with 50 J/m². Cells were analysed at various times following UV irradiation for the number of γH2AX-positive cells via immunofluorescence. Error bars represent SE; Student’s T test. *p < 0.05, **p < 0.01, ***p < 0.001