Normal mammary gland development in Stat3 depleted glands.

<p>(A) Whole mount staining of mammary glands of 5-week-old <i>Stat3^fl/fl;K14-Cre⁻</i> and <i>Stat3^fl/fl;K14-Cre⁺</i> females. (B) Flow cytometry analysis of luminal and basal cells isolated from mammary glands of <i>Stat3^fl/fl;K14-Cre⁻</i> and <i>Stat3^fl/fl;K14-Cre⁺</i> females four weeks after natural weaning. p value was determined using Student’s t test. ns: not significant.</p

<i>Stat3^fl/fl;BLG-Cre⁺</i> glands show incomplete involution and luminal progenitors have reduced proliferative capacity.

<p>(A) RT-PCR analysis of Stat3 expression in FACS sorted populations of mammary epithelial cells. MRU: mammary repopulating units. (B, C) H&E staining of sections of <i>Stat3^fl/fl;BLG-Cre⁻</i> and <i>Stat3^fl/fl;BLG-Cre⁺</i> mammary glands collected at day 5 of the second gestation (B) or four weeks after natural weaning (C). (D) Western blot analysis of four <i>Stat3^fl/fl;BLG-Cre⁻</i> and five <i>Stat3^fl/fl;BLG-Cre⁺</i> mammary glands four weeks after natural weaning for the expression or activation of Stat5, Erk, Akt, β-casein and WAP. β-actin was used as a loading control. (E) Immunohistochemistry staining for pStat5 (red) and E-cadherin (green) in mammary gland sections from <i>Stat3^fl/fl;BLG-Cre⁻</i> and <i>Stat3^fl/fl;BLG-Cre⁺</i> mice collected four weeks after natural weaning. Nuclei were stained with Hoechst 33342 (blue). (F) Flow cytometry analysis of luminal progenitors isolated from mammary glands of <i>Stat3^fl/fl;BLG-Cre⁻</i> and <i>Stat3^fl/fl;BLG-Cre⁺</i> females four weeks after natural weaning. (G) <i>In vitro</i> colony forming analysis performed on CD24⁺ CD49f^hi CD61⁺ luminal progenitor cells sorted from <i>Stat3^fl/fl;BLG-Cre⁻</i> and <i>Stat3^fl/fl;BLG-Cre⁺</i> mammary glands. Points represent the value for each mouse and lines depict mean values for each group. p value was determined using Student’s t test, * p<0.05.</p

BLG-Cre mediated deletion of Stat3 affects repopulating frequency of stem cells and outgrowth phenotype.

<p>(A) Whole mount staining of mammary outgrowths originating from CD24⁺ CD49f^hi basal cells sorted from mammary glands of <i>Stat3^fl/fl,BLG-Cre⁻</i> and <i>Stat3^fl/fl;BLG-Cre⁺</i> females four weeks after natural weaning. (B) Limiting dilution analysis of the repopulating frequency of the mammary stem cell-enriched population sorted from mammary glands of <i>Stat3</i>^fl/fl,BLG-Cre<i>⁻</i> and <i>Stat3</i>^fl/fl;BLG-Cre⁺ females four weeks after natural weaning. Number of outgrowths is shown per number of transplanted fat pads. CI: confidence interval.</p

Stat3 is required to maintain the multipotency of mammary stem cells and their proliferative potential.

<p>(A) Whole mount staining of mammary outgrowths originating from CD24⁺ CD49f^hi basal cells sorted from mammary glands of 5-week-old <i>Stat3^fl/fl,K14-Cre⁻</i> and <i>Stat3^fl/fl;K14-Cre⁺</i> females. (B) Limiting dilution analysis to assess the repopulating frequency of the mammary stem cell-enriched population sorted from mammary glands of 5-week-old <i>Stat3^fl/fl,K14-Cre⁻</i> and <i>Stat3^fl/fl;K14-Cre⁺</i> females. Number of outgrowths per number of transplanted fat pads and percentage of fat pad filled ± standard error of the mean are shown. CI: confidence interval. (C) H&E staining of mammary outgrowths originating from CD24⁺ CD49f^hi basal cells sorted from mammary glands of 5-week-old <i>Stat3^fl/fl;K14-Cre⁻</i> and <i>Stat3^fl/fl;K14-Cre⁺</i> females. (D, E) Immunohistochemistry staining for pStat5 (red, D), Ki67 (red, E) and E-cadherin (green) in mammary outgrowths originating from CD24⁺ CD49f^hi cells from mammary glands of 5-week-old <i>Stat3^fl/fl,K14-Cre⁻</i> and <i>Stat3^fl/fl;K14-Cre⁺</i> females. Nuclei were stained with Hoechst 33342 (blue).</p