Fig. 4

Abundance of myeloid-derived suppressor cells and M2-like macrophages in AI glands at the peak of cell death secreting CCL9. A. Temporal pattern of F4/80 positive cell distribution in AI and GI mammary glands harvested between days 7 and 25 PPM. FFPE sections of mammary glands were subjected to IHC using F4/80 antibody. Images of the stained slides were captured using Vectra 3.0 and analyzed using InForm software. n = 6 per group per time point. B. Mammary glands from AI-day 11 and GI-day 25 female mice were processed to identify different subpopulations of myeloid cells by multi-color flow cytometry. The representative flow cytometric dot plots and graphs of % B. CD45 + leukocytes; C. CD11b + Gr1 + MDSCs (out of CD45 + cells); D. CD11b + F4/80 + total macrophages (out of CD45 + cells); E. MHCII + M1-like macrophages (out of CD45 + CD11b + F4/80 + total macrophages); F. CD206 + M2-like macrophages (out of CD45 + CD11b + F4/80 + total macrophages); n = 3 per group. G. CD11b + Gr1 + MDSCs, and H. CD11b + F4/80 + MHCII-CD206 + M2-like macrophages were sorted from mammary glands of AI-day 11 and GI-day 25 mice. The cells were lysed and subjected to cytokine/chemokine array analysis. The images show expression of cytokine/chemokine and bar diagrams showing CCL9 expression quantified using ImageJ software. I. Quantitative RT-PCR analysis of Ccl9 expression in mammary glands over time (days 7 to 28 PPM), n = 3 per group. J. Quantitative RT-PCR analysis of Tgfβ3expression in mammary glands over time (days 7 to 28 PPM), n = 3 per group