Fig 2: Flow Cytometry Schema to Profile Adipose Stromal Cells and Validation by Ex Vivo Differentiation(A) Flow cytometry schema of adipose stromal cell types using combinations of cell surface markers, applied to all Cre;Rosa-EYFP adipose depots.(B) Representative flow cytometry plots of inguinal WAT live cells. (Left panel) Plot of live cells stained with CD45 (PE-Cy7) and CD11b (APC-Cy7). (Middle panel) Plot of CD45-CD11b- cells stained with SCA1 (Pacific Blue) and CD31 (APC). (Right panel) Histogram of EYFP fluorescence within APCs (Lin-SCA1+). Label above plots indicates parent populations.(C) iWAT APCs cultured in growth media (left) or adipogenic induction media (right), shown 6 days after induction. Scale bar represents 0.5 mm.See also Figure S1.

Fig 3: Mesenchymal adipocyte progenitors are recruited into the adult mammary epithelium during cellular expansion. a Short trace regimen in the tamoxifen (TAM)-inducible PdgfrαCreERT R26mTmG model. b Wholemount (left) and tissue sections immunostained for GFP (green), tdTomato (red) (middle) and GFP with EpCAM (red), PDGFRα (magenta) (right) isolated from an adult gland 3 days following TAM injection. c Flow cytometry quantification of GFP+ cells within stromal (S), basal (B), luminal (L), and luminal Sca1+, Sca1- subsets in the 3d short trace (n = 3 mice). d Three week trace of PDGFRα+ cell progeny after TAM induction and hormone stimulation. e Native fluorescence within lobuloalveoli after E+P treatment (left) and GFP+ cells in tissue sections (middle, right). f GFP+ cells in mammary subsets after a 3 week trace (n = 3 mice). g PDGFRα+ cell tracing during pregnancy. h Wholemount fluorescence showing GFP+ lobuloalveolar structures during mid-pregnancy (left) and immunostaining on tissue sections (middle, right). i GFP+ cell proportions during pregnancy (n = 3 mice). j Lineage tracing Pref-1+ mesenchymal adipocyte progenitors in the Pref-1-rtTA TRE-Cre R26mTmG model following DOX induction and hormone stimulation. k Mammary tissue sections from Pref-1 tracing mice co-stained for eGFP (green) and tdTomato (red) (left), eGFP with epithelial markers EpCAM (red) (middle) and K19 (red), K14 (magenta) (right). l GFP+ cell distribution in mammary subsets of Pref-1 adult mice stimulated with hormones (n = 3 mice). m Mature adipocyte lineage tracing model subjected to TAM induction and pregnancy. n Mammary tissue isolated from adipocyte tracing mice analyzed by wholemount (left) and immunostaining (middle, right) with eGFP (green), tdTomato (red), PDGFRα+(magenta) and DAPI (blue), showing an absence of mature adipocyte recruitment into the epithelium (n = 3 mice). Scale bar = 50 µm for wholemounts and 25 µm for tissue sections; arrowheads indicate GFP+ cells. Images are representative of 5 fields per tissue section per mouse. White asterisk denotes associated Supplementary Movies 2 and 3. Data represent mean ± s.e.m. Source data are provided as a Source Data file

Fig 4: Group A Streptococcus (GAS) infection depletes bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) 24 hr post infection.(A) Experimental time frame of GAS infection and BM analysis. (B) Flow plot of HSPC gating and representation of different surface expression of cKit and Sca-1 during infection. Plots are gated from lineage negative BM cells. (C–F) Absolute number of HSPCs, hematopoietic stem cells (HSCs), multipotent progenitors (MPP) 3, and MPP4 in the BM of naïve and GAS-infected mice. Spleen populations of (G) HSPCs, (H) HSC/MPP1, (I) MPP2, and (J) MMP3/4 identified by differential expression of CD150 and CD48. (C–J) Data is representative of three independent experiments; n = 4–5 mice/group; statistical comparison done using unpaired t-test; *p < 0.05, **p < 0.01. Outliers were identified using the ROUT method (Q = 5%). Figure 2—source data 1.BM counts of HSPC populations of naïve and infected mice and spleen HSPC populations.

Fig 5: Klf4 Inhibits Proliferation and Self-Renewal of TICs(A) Expression of Sca-1 and CD49f by the Akt cell line (right); cells with control antibodies (left).(B) Sphere-forming ability of the Akt cell line (3,000 cells/well) in Matrigel after infection with control (shCon) or shKlf4 lentivirus. Scale bars, 30 μm. Experiments were repeated 3 times. Data represent means ± SDs. p = 0.001.(C) Spheres from shKlf4 and shCon Akt cell line were dissociated, and equal numbers of cells (2,000) were passaged 6 times in triplicate in Matrigel. The sphere number diminished with passage as cells became more migratory and infiltrated the Matrigel (bottom). Scale bars, 30 μm.(D) Sphere-forming ability of the Akt cell line (3,000 cells/well) in Matrigel after infection with control or Klf4-expressing constructs. Scale bars, 30 μm. Experiments were repeated 3 times. Data represent means ± SDs. p < 0.006.