Fig 2: Deletion of Sox9 results in mammary gland defects in 3 and 5 weeks old mice. Whole mount analysis of mammary glands from control (MMTV-Cre; Sox9wt/wt) and Sox9 cKO (MMTV-Cre; Sox9fl/fl) mice. Mammary glands were harvested at 3, 5, 8 weeks, mid-pregnancy, lactation day 1, and lactation day 10 and stained with carmine alum and analyzed under the microscope.

Fig 3: Long-term linage tracing shows that Sox9 expressing cells are capable of giving rise to both luminal and myoepithelial lineages (A). X-gal stained tissue section of adult virgin Sox9-CreERT2; R26R-LacZ mammary gland injected with TM and harvested after 4 weeks. Arrows point to reporter activity in myoepithelial cells. (B). GFP stained tissue section of adult virgin Sox9-CreERT2; CAG-EGFP mammary gland injected with TM and harvested after 4 weeks. Arrows highlight the progeny of Sox9 expressing cells within the myoepithelial layer (C). Flow cytometry analysis of CD24, GFP expression of GFP positive and negative cells isolated from above adult virgin mice mammary glands. (D). Flow cytometry analysis of CD61, CD29 expression of GFP+ cells isolated from above adult virgin mice mammary glands.

Fig 4: Sox9 deleted luminal cells are lost over time. (A). LacZ staining of whole mount mammary glands from 5 week old Sox9 cKO (R26R; MMTV-Cre; Sox9fl/fl) mouse. (B). GFP in whole mount 8 week old Sox9 cKO (CAG-EGFP; MMTV-Cre; Sox9fl/fl) was visualized with fluorescence microscope. (C). Section of mammary glands from 5-week old Sox9 cKO (R26R; MMTV-Cre; Sox9fl/fl) mouse, stained with X-gal. (D). Section of mammary glands from 8 week old Sox9 cKO (CAG-EGFP; MMTV-Cre; Sox9fl/fl) mouse, immuno-labeling with anti-GFP (green) and anti-a-SMA (red) antibodies. (E). CD24, GFP FACS analysis of cells isolated from control (CAG-EGFP;MMTV-Cre; Sox9wt/wt) mouse mammary glands. (F). CD24, GFP Flow cytometry analysis of cells isolated from Sox9 deletion (MMTV-Cre; Sox9fl/fl; CAG-EGFP) mouse mammary glands.

Fig 5: Short-term linage tracing shows that Sox9 is exclusively expressed in luminal cells. (A). Sox9-CreERT2; CAG-EGFP mice were injected with tamoxifen. 72 hr after injection, the mice were sacrificed for mammary gland staining and FACS analysis. Sections of the mouse mammary gland were stained with GFP (green) and myoepithelial cell marker (a-SMA) (red) antibodies and DAPI (staining nuclei, blue). (B) Flow cytometry analysis of CD24 expression of GFP-positive and -negative cells isolated from above tamoxifen treated mouse mammary glands. (C). Flow cytometry analysis of all CD24+ luminal cells (both GFP+ and GFP-) isolated from the above tamoxifen treated mouse mammary glands using CD61 and CD49f expression. This demonstrates that luminal cells of the mammary gland are comprised of CD61+ luminal progenitors and CD61- differentiated luminal cells. (D). CD61, CD49f flow cytometry analysis of only GFP+ cells isolated from above tamoxifen treated mouse mammary glands shows that GFP+ cells are mostly CD61+ luminal progenitors. (E) CD24, CD29 flow cytometry analysis of GFP positive cells isolated from above tamoxifen treated mouse mammary glands.