PMC

Decreased colony formation in C/EBPβ-null MECs. Colony forming ability on cultured feeder layers was significantly decreased in C/EBPβ^−/−;CFP⁺ MECs as compared to wildtype (C/EBPβ^+/+;CFP⁺). Data represent mean ± SEM of 3 independent experiments (*p<0.0001). Images depict crystal violet-stained colonies in wildtype and C/EBPβ^−/−;CFP⁺ cells, and insets demonstrate a single CFP⁺ colony. Scale bars, 5 mm (large panels) and 250 μm (insets).

Decreased mammosphere formation in C/EBPβ-null MECs. Graphs illustrate the number of secondary mammospheres formed per 5000 cells expressed as mammosphere efficiency using either (A) conditional wildtype (C/EBPβ^+/+;R26R) and C/EBPβ^fl/fl;R26R cells transduced with Ad.Cre1 or (B) germline wildtype (C/EBPβ^+/+) and C/EBPβ^−/− MECs. The ability of C/EBPβ-null cells to form secondary mammospheres was significantly inhibited using both these models (*p<0.0001). Data represent mean ± SEM of 1 representative experiment and the experiment was performed 3 times.

Decreased MRUs in C/EBPβ-null outgrowths when transplanted at limiting dilution. MECs were isolated from wildtype (C/EBPβ^+/+;R26R) and C/EBPβ^fl/fl;R26R glands, transduced with Ad.Cre1 and transplanted into syngeneic hosts at limiting dilutions. (A) Outgrowth potential was significantly decreased in C/EBPβ-deleted glands as compared to wildtype (p=0.017). The ability of C/EBPβ-null MECs to fill the fat pad was also significantly impaired as compared to wildtype (p=0.0005). Photomicrographs depict whole mount analysis of glands injected with 250 of wildtype (B) or C/EBPβ-null (C) cells. Scale bars, 5 mm.

Altered stem/progenitor cell populations in C/EBPβ^−/− mammary glands. (A) Dot plots depict that the percentage of LIN⁻CD24⁺CD29^hi cells, which contain MRUs, is decreased in C/EBPβ^−/− MECs, while the LIN⁻CD24^hiCD29^lo subpopulation is increased. (B) The LIN⁻CD24^hiCD29^lo subpopulation was gated and CD61 expression was examined within this subpopulation. C/EBPβ^−/− MECs express a lower percentage of LIN⁻CD24^hiCD29^loCD61⁺ cells as compared to wildtype (C/EBPβ^+/+). (C) The LIN⁻CD24^hiSca1^hi subpopulation is increased in C/EBPβ^−/− glands as compared to wildtype, which is accompanied by the loss of LIN⁻CD24^loSca1⁻ cells. For all FACS plots, lineage-positive cells were excluded using a mouse lineage panel kit plus biotin-conjugated CD31 and CD140a antibodies. Each dot/contour plot represents 1 experiment, and bar graphs depict the mean ± SEM of 4 independent experiments (*p<0.0001).

Decreased outgrowth of C/EBPβ^−/− serial transplants. CFP⁺ epithelia were dissected from wildtype (C/EBPβ^+/+;CFP⁺) or germline C/EBPβ^−/−; CFP⁺ glands and transplanted sequentially for 3 consecutive generations, harvesting tissue at each generation after 8 weeks of outgrowth. (A) While take rate remained similar between wildtype and C/EBPβ^−/−; CFP⁺ tissue, the ability to reconstitute the fat pad significantly decreased with subsequent generations (p=0.002) in C/EBPβ-null glands as compared to wildtype (Gen 2 p=0.003, Gen 3 p=0.004). (B) Fluorescent micrographs (inverted images) depict representative images of generation 3 outgrowths of wildtype and C/EBPβ^−/−; CFP⁺ glands in mature virgin mice (top) or late pregnant (P19) glands (bottom). (C) Images depict generation 4 outgrowths from wildtype and stunted C/EBPβ^−/− CFP⁺ donor glands. The number of outgrowths represented by each micrograph is depicted below each image. Scale bars, 5 mm.

Conditional deletion of C/EBPβ results in altered ductal morphogenesis and decreased lobuloalveolar development. MECs were isolated from 10-week old wildtype (C/EBPβ^+/+;R26R) and C/EBPβ^fl/fl;R26R mice, transduced with Ad.Cre1 in vitro and Cre-mediated recombination was examined (n=3). (A) qPCR for C/EBPβ showed that the expression of C/EBPβ was decreased >300-fold in transduced C/EBPβ^fl/fl;R26R MECs as compared to wildtype. (B–G) Transduced cells were transplanted into syngeneic hosts and stained with X-gal after 10 weeks of outgrowth. Large, dilated ducts were evident in virgin C/EBPβ-deleted glands as compared to wildtype by whole mount analysis (B,C) and H&E staining of paraffin-embedded sections (D,E). H&E staining of pregnant glands (P16–17) illustrated decreased alveolar development in C/EBPβ-null glands (G) as compared to wildtype (F), the latter of which contained lipid droplets. Uniform lacZ expression was observed throughout the mammary gland, indicative of a high extent of Cre-mediated recombination. Scale bars, 5 mm (B,C) or 20μm (D–G).