Fig. 6. Depletion of macrophages or genetic knockout of Notch signaling in macrophage reduces stem cell activity of Dll1⁺ basal cells.

(A) Table shows take rate of transplantation with 200 P4-Dll1⁺ and P4-Dll1⁻ cells from Dll1-mCherry mouse mammary gland. Recipient mice were treated with IgG (control) and Csf1r antibody (500 μg/mouse) for 4–5 weeks. (B) Representative alum carmine stained whole-mount mammary outgrowths from (A). (C-D) Quantification of macrophages from mammary gland and peripheral blood from control and Csf1r antibody treated mice. FACS was performed using CD45 and F4/80 antibodies to detect macrophages (n= 5 mice per condition). (E) Table shows take rate of transplantation with 500 P4-Dll1⁺ and P4-Dll1⁻ cells from Dll1-mCherry mice mammary gland into MaFIA recipient mice. Recipient MaFIA mice were treated with either vehicle or AP20187 (5 mg/Kg) for 3 weeks. AP20187 was used to induce depletion of macrophages in MaFIA mice. (F) Representative alum carmine stained whole-mount mammary outgrowths from (E). (G-H) Quantification of macrophages from mammary gland and peripheral blood from control and MaFIA treated mice. FACS was performed using CD45 and F4/80 antibodies to detect macrophages (n= 5 mice per condition). (I) Representative alum carmine stained whole-mount mammary outgrowths from WT and Rbpjk^cKO (CD11c-Cre; Rbpjk^f/f) mice at 5–6 weeks. CD11c-Cre deletes Rbpjk in mammary tissue macrophages (41). (J-L) Box plot analyses of ductal elongation, branching and terminal end buds (TEB) counts in WT and Rbpjk^cKO mice. Quantification of ductal branching (tertiary branch points) was measured in defined area. (M) Box plot showing percentage of P4 (basal) cells in WT and Rpjk^cKO mouse after FACS using CD24 and CD29 antibodies in Lin⁻ cells. In (J-M), n = 5 samples for WT and Rbpjk^cKO. (N) Table shows take rate of transplantation using mixed population of 500 P4-Dll1⁺ and 2000 mammary macrophages. Mammary macrophages were sorted from Actin-GFP mice using F4/80 and CD140 antibodies and either infected with control lentivirus or Rbpjk shRNAs (KD1 and KD2). P4-Dll1⁺ cells were obtained from sorting of Dll1-mCherry mouse mammary gland. p=0.0266 by Fisher Exact test when comparing the two control groups vs. the two Rbjp KD groups. Please refer to schematic in fig. S9H for additional details of the experimental design. (O) Representative carmine alum stained images of transplants of different groups from (N). In all box plots, the boxes represent the 75th, 50th and 25th percentile of the values. The top and bottom lines represent maximum and minimal data points within the 1.5x IQ (inter quarter) range, respectively. Mann Whitney U test was used in (C), (D), (G), (H) and (J-M). Size bar, 1 mm in (B), (F) and (O), 2 mm in (I).