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1.
Figure 5

Figure 5. From: TBX3 over-expression causes mammary gland hyperplasia and increases mammary stem-like cells in an inducible transgenic mouse model.

TBX3 over-expression is associated with increased mammary stem-like cell population. A and B) FACS analysis of mammary stem-like cells. (A) Representative graphs of gating strategies used to select Lin- from Lin+ cell population. Anti-CD31, anti-CD45 and anti-TER119 antibodies were used to separate the Lin- and Lin+ cells from harvested mammary glands of 12-week old female virgin mice (doxycycline induced and un-induced double transgenic littermates) (left panel). Gating strategy used to purify CD29highCD24+ cells from Lin- cell population (right panel). B) Statistical analysis of the average frequency of mammary stem-like cells (CD29high, CD24+) in Lin- population. Two mice from each group were used to generate the data. Error bars indicate SD; n = 2, *, P < 0.05.

Jing Liu, et al. BMC Dev Biol. 2011;11:65-65.
2.
Figure 3

Figure 3. From: TBX3 over-expression causes mammary gland hyperplasia and increases mammary stem-like cells in an inducible transgenic mouse model.

TBX3 over-expression promotes cell proliferation. A) Edu cell proliferation assay. The fourth mammary glands from doxycycline induced and un-induced double transgenic mice were harvested at 10.5 dpc and 5μm thick sections were embedded in paraffin and subjected to the Click-iT EdU proliferation Assay (Promega). EdU that had been incorporated into newly synthesized DNA was detected by Alexa Fluor 594 azide (red) and cell nuclei were stained with Hoechst 33342 (blue). All Images were captured at 20× magnification. B) Quantification of Edu positive cells. Fifteen random 20× fields were taken from each group of litter matched doxycycline induced and un-induced double transgenic mice. The proliferating cells were quantified and normalized to the total cell number in each field. The graph is generated from the average ratio (Edu/Hoechst) of fifteen 20× fields in each group. Error bars indicate SD; n = 1. **,P < 0.001.

Jing Liu, et al. BMC Dev Biol. 2011;11:65-65.
3.
Figure 4

Figure 4. From: TBX3 over-expression causes mammary gland hyperplasia and increases mammary stem-like cells in an inducible transgenic mouse model.

TBX3 inhibits NFKBIB promoter activity. A) Luciferase reporter assay. COS-7 cells were transfected with either pcDNA3.1-Myc (control) or pcDNA3.1-Myc-TBX3 expression vectors together with the pGL3-NFκBIB luciferase reporter construct and a β-galactosidase control plasmid (pcDNA3.1/His/LacZ). Cell lysates were harvested 48 hours after transfection and used to measure luciferase activity. β-galactosidase enzyme activity was also measured and used to normalize luciferase activity. Error bars indicate SD; n = 3. **, P < 0.001. B) Immunohistochemical staining of Nfκbib in doxycycline induced and un-induced double transgenic mice at 10 weeks of age. The third mammary glands were fixed and embedded in paraffin. Five micrometer thick sections were blocked with hydrogen peroxide and incubated with rabbit anti-Nfκbib antibody overnight. Biotinylated goat anti-rabbit IgG was used as a secondary antibody. Standard ABC kit and DAB kit were used for visualization. Nfκbib was down-regulated in the doxycycline induced double transgenic mouse as compared to its un-induced double transgenic littermate control. Images were captured at 40× magnification.

Jing Liu, et al. BMC Dev Biol. 2011;11:65-65.
4.
Figure 1

Figure 1. From: TBX3 over-expression causes mammary gland hyperplasia and increases mammary stem-like cells in an inducible transgenic mouse model.

TBX3 transgene expression is tightly controlled by doxycycline administration within mammary glands of double-transgenic mice. A) Diagram of tetracycline inducible TBX3 transgene cassette. In the presence of doxycycline, the tetracycline reverse transcriptional activator (rtTA), expressed in mammary epithelium, binds to the tetracycline-operator (Tet-On), which in turn activates the transcription of TBX3 and firefly luciferase. B) Left panel: In vivo detection of luciferase reporter activity. Fifteen-week old doxycycline induced and un-induced double transgenic female mice were injected with an aqueous solution of luciferin into the peritoneal cavity. Images were taken with an ICCD camera five minutes later. Right panel: Luciferase reporter activity in tet-TBX3; MMTV-rtTA transgenic mammary glands. Mammary glands from doxycycline induced and un-induced double transgenic mice were dissected and lysates were prepared. The luciferase activity of 10μl of total protein lysate was measured. Numbers on X-axis indicate the pair of mammary gland used in the assay (n = 1). C) Immunohistochemical analysis of TBX3 expression in doxycycline induced and un-induced double transgenic littermates at 10 weeks of age. Images were captured at 40× magnification.

Jing Liu, et al. BMC Dev Biol. 2011;11:65-65.
5.
Figure 2

Figure 2. From: TBX3 over-expression causes mammary gland hyperplasia and increases mammary stem-like cells in an inducible transgenic mouse model.

TBX3 over-expression causes accelerated mammary gland development and hyperplasia. A) Whole mount analysis of the 4th mammary gland from 7-weeks, 10-weeks, 12-weeks old virgin mice and day 10.5 dpc pregnant mice. Acceleration of mammary gland development is observed in doxycycline induced double transgenic mice from 7 to 12 weeks of age. TEBs are present in un-induced control mice at 7- and 10- weeks of age (arrows), whereas TEBs in doxycycline induced double transgenic mice pass the fat pad and start to disappear. At all time points, branching morphogenesis was promoted in the doxycycline induced double transgenic mice. Representative whole mount of the 4th mammary gland from 10.5 dpc pregnant un-induced double transgenic mouse has less alveoli tissue than the 4th mammary gland from day 10.5 dpc pregnant doxycycline induced double transgenic mouse (n = 3). All images were captured at 5× magnification. Image insets were captured at 20× magnification. B) Whole mount analysis of the 1st mammary gland from un-induced double transgenic mice and doxycycline induced double transgenic mice at 12 weeks of age and pregnant 10.5 dpc. Mammary glands from doxycycline induced double transgenic mice displayed enhanced side branching and advanced development of alveolar structure. Images were captured at 10× magnification. Hematoxylin and eosin stain performed on the 3rd mammary gland of doxycycline induced and un-induced double transgenic mice at 15 weeks of age showed mild hyperplasia (arrows) and disturbed organization in the mammary epithelium (arrowheads) (n = 3). Images were captured at 40× magnification.

Jing Liu, et al. BMC Dev Biol. 2011;11:65-65.

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